Regulation of epithelial tissue by hedgehog-like polypeptides, and formulations and uses related thereto

ABSTRACT

The invention provides methods and compositions for modulating hair growth.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/955,552,filed Oct. 20, 1997, and now abandoned, the specification of which isincorporated by reference herein.

BACKGROUND OF THE INVENTION

Pattern formation is the activity by which embryonic cells form orderedspatial arrangements of differentiated tissues. The physical complexityof higher organisms arises during embryogenesis through the interplay ofcell-intrinsic lineage and cell-extrinsic signaling. Inductiveinteractions are essential to embryonic patterning in vertebratedevelopment from the earliest establishment of the body plan, to thepatterning of the organ systems, to the generation of diverse cell typesduring tissue differentiation (Davidson, E., (1990) Development 108:365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al.,(1992) Cell 68: 257-270). The effects of developmental cell interactionsare varied. Typically, responding cells are diverted from one route ofcell differentiation to another by inducing cells that differ from boththe uninduced and induced states of the responding cells (inductions).Sometimes cells induce their neighbors to differentiate like themselves(homoiogenetic induction); in other cases a cell inhibits its neighborsfrom differentiating like itself. Cell interactions in early developmentmay be sequential, such that an initial induction between two cell typesleads to a progressive amplification of diversity. Moreover, inductiveinteractions occur not only in embryos, but in adult cells as well, andcan act to establish and maintain morphogenetic patterns as well asinduce differentiation (J. B. Gurdon (1992) Cell 68:185-199).

Members of the Hedgehog family of signaling molecules mediate manyimportant short- and long-range patterning processes during invertebrateand vertebrate development. In the fly a single hedgehog gene regulatessegmental and imaginal disc patterning. In contrast, in vertebrates ahedgehog gene family is involved in the control of left-right asymmetry,polarity in the CNS, somites and limb, organogenesis, chondrogenesis andspermatogenesis.

The first hedgehog gene was identified by a genetic screen in thefruitfly Drosophila melanogaster (Nüsslein-Volhard, C. and Wieschaus, E.(1980) Nature 287, 795-801). This screen identified a number ofmutations affecting embryonic and larval development. In 1992 and 1993,the molecular nature of the Drosophila hedgehog (hh) gene was reported(C. F., Lee et al. (1992) Cell 71, 33-50), and since then, severalhedgehog homologues have been isolated from various vertebrate species.While only one hedgehog gene has been found in Drosophila and otherinvertebrates, multiple Hedgehog genes are present in vertebrates.

The various Hedgehog proteins consist of a signal peptide, a highlyconserved N-terminal region, and a more divergent C-terminal domain. Inaddition to signal sequence cleavage in the secretory pathway (Lee, J.J. et al. (1992) Cell 71:33-50; Tabata, T. et al. (1992) Genes Dev.2635-2645; Chang, D. E. et al. (1994) Development 120:3339-3353),Hedgehog precursor proteins undergo an internal autoproteolytic cleavagewhich depends on conserved sequences in the C-terminal portion (Lee etal. (1994) Science 266:1528-1537; Porter et al (1995) Nature374:363-366). This autocleavage leads to a 19 kD N-terminal peptide anda C-terminal peptide of 26-28 kD (Lee et al. (1992) supra; Tabata et al.(1992) supra; Chang et al. (1994) supra; Lee et al. (1994) supra;Bumcrot, D. A., et al. (1995) Mol. Cell. Biol. 15:2294-2303; Porter etal. (1995) supra; Ekker, S. C. et al. (1995) Curr. Biol. 5:944-955; Lai,C. J. et al. (1995) Development 121:2349-2360). The N-terminal peptidestays tightly associated with the surface of cells in which it wassynthesized, while the C-terminal peptide is freely diffusible both invitro and in vivo (Lee et al. (1994) supra; Bumcrot et al. (1995) supra;Mart′, E. et al. (1995) Development 121:2537-2547; Roelink, H. et al.(1995) Cell 81:445-455). Interestingly, cell surface retention of theN-terminal peptide is dependent on autocleavage, as a truncated form ofHH encoded by an RNA which terminates precisely at the normal positionof internal cleavage is diffusible in vitro (Porter et al. (1995) supra)and in vivo (Porter, J. A. et al. (1996) Cell 86, 21-34). Biochemicalstudies have shown that the autoproteolytic cleavage of the HH precursorprotein proceeds through an internal thioester intermediate whichsubsequently is cleaved in a nucleophilic substitution. It is likelythat the nucleophile is a small lipophilic molecule which becomescovalently bound to the C-terminal end of the N-peptide (Porter et al.(1996) supra), tethering it to the cell surface. The biologicalimplications are profound. As a result of the tethering, a high localconcentration of N-terminal Hedgehog peptide is generated on the surfaceof the Hedgehog producing cells. It is this N-terminal peptide which isboth necessary and sufficient for short and long range Hedgehogsignaling activities in Drosophila and vertebrates (Porter et al. (1995)supra; Ekker et al. (1995) supra Lai et al. (1995) supra; Roelink, H. etal. (1995) Cell 81:445-455; Porter et al. (1996) supra; Fietz, M. J. etal. (1995) Curr. Biol. 5:643-651; Fan, C. -M. et al. (1995) Cell81:457-465; Mart′, E., et al. (1995) Nature 375:322-325; Lopez-Martinezet al. (1995) Curr. Biol 5:791-795; Ekker. S. C. et al. (1995)Development 121:2337-2347; Forbes, A. J. et al.(1996) Development122:1125-1135).

HH has been implicated in short- and longe range patterning processes atvarious sites during Drosophila development. In the establishment ofsegment polarity in early embryos, it has short range effects whichappear to be directly mediated, while in the patterning of the imaginaldiscs, it induces long range effects via the induction of secondarysignals.

In vertebrates, several hedgehog genes have been cloned in the past fewyears (see Table 1). Of these genes, Shh has received most of theexperimental attention, as it is expressed in different organizingcenters which are the sources of signals that pattern neighbouringtissues. Recent evidence indicates that Shh is involved in theseinteractions.

The interaction of a hedgehog protein with one of its cognate receptor,patched, sets in motion a cascade involving the activation andinhibition of downstream effectors, the ultimate consequence of whichis, in some instances, a detectable change in the transcription ortranslation of a gene. Transcriptional targets of hedgehog signaling arethe patched gene itself (Hidalgo and Ingham, 1990 Development 110,291-301; Marigo et al., 1996) and the vertebrate homologs of thedrosophila cubitus interruptus (Ci) gene, the GLI genes (Hui et al.(1994) Dev Biol 162:402-413). Patched gene expression has been shown tobe induced in cells of the limb bud and the neural plate that areresponsive to Shh. (Marigo et al. (1996) Development 122:1225-1233). TheGLI genes encode putative transcription factors having zinc finger DNAbinding domains (Orenic et al. (1990) Genes & Dev 4:1053-1067; Kinzleret al. (1990) Mol Cell Biol 10:634-642). Transcription of the GLI genehas been reported to be upregulated in response to hedgehog in limbbuds, while transcription of the GLI3 gene is downregulated in responseto hedgehog induction (Marigo et al. (1996) Development 122:1225-1233).Moreover, it has been demonstrated that elevated levels of Ci aresufficient to activate patched (ptc) and other hedgehog target genes,even in the absence of hedgehog activity.

SUMMARY OF THE INVENTION

One aspect of the present application relates to a method for modulatingthe growth state of an epithelial cell by ectopically contacting theepithelial cell, in vitro or in vivo, with a hedgehog therapeutic or ptctherapeutic in an amount effective to alter the rate (promote orinhibit) of proliferation of the epithelial cell, e.g., relative to theabsence of administeration of the hedgehog therapeutic or ptctherapeutic. The subject method can be used, for example, to modulatethe growth state of an epithelial tissue, such as for inducing theformation of skin or other cutaneous tissue, or for inducing growth ofhair.

Wherein the subject method is carried out using a hedgehog therapeutic,the hedgehog therapeutic preferably a polypeptide including a hedgehogportion comprising at least a bioactive extracellular portion of ahedgehog protein, e.g., the hedgehog portion includes at least 50, 100or 150 (contiguous) amino acid residues of an N-terminal half of ahedgehog protein. In preferred embodiments, the hedgehog portionincludes at least a portion of the hedgehog protein corresponding to a19 kd fragment of the extracellular domain of a hedgehog protein.

In certain preferred embodiments, the hedgehog portion has an amino acidsequence at least 60, 75, 85. or 95 percent identical with a hedgehogprotein of any of SEQ ID Nos. 10-18 or 20, though sequences identical tothose sequence listing entries are also contemplated as useful in thepresent method. The hedgehog portion can be encoded by a nucleic acidwhich hybridizes under stringent conditions to a nucleic acid sequenceof any of SEQ ID Nos. 1-9 or 19, e.g., the hedgehog portion can beencoded by a vertebrate hedgehog gene, especially a human hedgehog gene.

In certain embodiments, the hedgehog polypeptide is modified with one ormore sterol moieties, e.g., cholesterol or a derivative thereof.

In certain embodiments, the hedgehog polypeptide is modified with one ormore fatty acid moieties, such as a fatty acid moiety selected from thegroup consisting of myristoyl, palmitoyl, stearoyl, and arachidoyl.

In certain embodiments, the hedgehog polypeptide is modified with one ormore aromatic hydrocarbons, such as benzene, perylene, phenanthrene,anthracene, naphthalene, pyrene, chrysene, or naphthacene.

In certain embodiments, the hedgehog polypeptide is modified one or moretimes with a C7 -C30 alkyl or cycloalkyl.

In other embodiments, the subject method can be carried out byadministering a gene activation construct, wherein the gene activationconstruct is deigned to recombine with a genomic hedgehog gene of thepatient to provide a heterologous transcriptional regulatory sequenceoperatively linked to a coding sequence of the hedgehog gene.

In still other embodiments, the subject method can be practiced with theadministration of a gene therapy construct encoding a hedgehogpolypeptide. For instance, the gene therapy construct can be provided ina composition selected from a group consisting of a recombinant viralparticle, a liposome, and a poly-cationic nucleic acid binding agent,

In yet other embodiments, the subject method can be carried out using aptc therapeutic. An exemplary ptc therapeutic is a small organicmolecule which binds to a patched protein and derepressespatched-mediated inhibition of mitosis, e.g., a molecule which binds topatched and mimics hedgehog-mediated patched signal transduction, whichbinds to patched and regulates patched-dependent gene expression. Forinstance, the binding of the ptc therapeutic to patched may result inupregulation of patched and/or gli expression.

In a more generic sense, the ptc therapeutic can be a small organicmolecule which interacts with epithelial cells to inducehedgehog-mediated patched signal transduction, such as by altering thelocalization, protein—protein binding and/or enzymatic activity of anintracellular protein involved in a patched signal pathway. Forinstance, the ptc therapeutic may alter the level of expression of ahedgehog protein, a patched protein or a protein involved in theintracellular signal transduction pathway of patched.

In certain embodiments, the ptc therapeutic is an antisense constructwhich inhibits the expression of a protein which is involved in thesignal transduction pathway of patched and the expression of whichantagonizes hedgehog-mediated signals. The antisense construct isperferably an oligonucleotide of about 20-30 nucleotides in length andhaving a GC content of at least 50 percent.

In other embodiments, the ptc therapeutic is an inhibitor of proteinkinase A (PKA), such as a 5-isoquinolinesulfonamide. The PKA inhibitorcan be a cyclic AMP analog. Exemplary PKA inhibitors includeN-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide.1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, KT5720, 8-bromo-cAMP,dibutyryl-cAMP and PKA Heat Stable Inhibitor isoform α. Anotherexemplary PKA inhibitor is represented in the general formula:

wherein,

R₁ and R₂ each can independently represent hydrogen, and as valence andstability permit a lower alkyl a lower alkenyl, a lower alkynyl, acarbonyl (such as a carboxyl, an ester, a formate, or a ketone), athiocarbonyl (such as a thioester, a thioacetate, or a thioformate), anamino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, asulfonate, a sulfonamido, —(CH₂)_(m)—R₈, —(CH₂)_(m)—OH.—(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₈, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₈, or

R₁ and R₂ taken together with N form a heterocycle (substituted orunsubstituted);

R₃ is absent or represents one or more substitutions to the isoquinolinering such as a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl(such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl(such as a thioester, a thioacetate, or a thioformate), an amino, anacylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate,a sulfonamido, —(CH₂)_(m)—R₈, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₈, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₈;

R₈ represents a substituted or unsubstituted aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle; and

n and m are independently for each occurrence zero or an integer in therange of 1 to 6.

The subject method can be used to treat, e.g., a epithelial disorder,such as in the control of a wound healing process. For instance, thesubect method can be used as part of such treatments as burn treatment,skin regeneration, skin grafting, pressure sore treatment, dermal ulcertreatment, post surgery scar reduction and treatment of ulcerativecolitis. In the control of hair growth, the subject method can used aspart of a treatment of alopecia.

Yet another aspect of the present invention concerns preparations of ahedgehog or ptc therapeutic formulated for topical application toepithelial tissue, e.g., to skin. For example, such formulations mayinclude a polypeptide comprising a hedgehog polypeptide sequenceincluding a bioactive fragment of a hedgehog protein, which polypeptideis formulated for topical application to epithelial tissue.

DETAILED DESCRIPTION OF THE INVENTION

FIGS. 1A, B and C illustrate the induction of hair growth on micetreated with various hedgehog formulations.

DETAILED DESCRIPTION OF THE INVENTION

Normal skin epidermis is a complex epithelial tissue containingkeratinocytes that are proliferating, differentiating and desquamating,and is stratified such that morphological and functional changes in thekeratinocytes occur in an orderly progression. The normal epidermis ismaintained in a dynamic steady state as proliferation of keratinocytescontinually compensates for the loss of cells which are shed from thesurface of the skin. Within the epidermis, proliferation takes place inthe basal layer of keratinocytes that are attached to the underlyingbasement membrane, and cells undergo terminal differentiation as theymigrate through the suprabasal layers, finally being shed from thetissue surface as dead, cornified squames. Three subpopulations of basalkeratinocytes have been defined by cell kinetic analysis: stem cells,transit-amplifying cells, and committed cells. Stem cells retain a highcapacity for self-renewal throughout adult life and are ultimatelyresponsible for epidermal maintenance and repair. The progeny of stemcells can either be stem cells themselves or cells known astransit-amplifying cells. Transit-amplifying cells divide a small numberof times, but have a high probability of producing daughters thatwithdraw irreversibly from the cell cycle and are committed todifferentiate terminally.

I. Overview

The present application is directed to the discovery that preparationsof hedgehog polypeptides can be used to control the formation and/ormaintenance of epithelial tissue. As described in the appended examples,hedgehog proteins are implicated in the proliferation of epithelial stemcells and may provide early signals that regulate the differentiation ofthe stem cells into epithelial tissues. In general, the method of thepresent invention comprises contacting an epithelial cell with an amountof a hedgehog therapeutic (defined infra) which produces a non-toxicresponse by the cell of (i) induction of epithelial tissue formation or(ii) inhibition of epithelial tissue formation, depending on the whetherthe hedgehog therapeutic is a sufficient hedgehog agonist or hedgehogantagonist. The subject method can be carried out on epithelial cellswhich may be either dispersed in culture or a part of an intact tissueor organ. Moreover, the method can be performed on cells which areprovided in culture (in vitro), or on cells in a whole animal (in vivo).

In one aspect, the present invention provides pharmaceuticalpreparations and methods for controlling the proliferation ofepithelially-derived tissue utilizing, as an active ingredient, ahedgehog polypeptide or a mimetic thereof. The invention also relates tomethods of controlling proliferation of epithelial-derived tissue by useof the pharmaceutical preparations of the invention.

The hedgehog formulations of the present invention may be used as partof regimens in the treatment of disorders of, or surgical or cosmeticrepair of, such epithelial tissues as skin and skin organs; corneal,lens and other ocular tissue; mucosal membranes; and periodontalepithelium. The methods and compositions disclosed herein provide forthe treatment or prevention of a variety of damaged epithelial andmucosal tissues. For instance, the subject method can be used to controlwound healing processes, as for example may be desirable in connectionwith any surgery involving epithelial tissue, such as fromdermatological or periodontal surgeries. Exemplary surgical repair forwhich hedgehog therapy is a candidate treatment include severe burn andskin regeneration, skin grafts, pressure sores, dermal ulcers, fissures,post surgery scar reduction, and ulcerative colitis.

In another aspect of the present invention, hedgehog preparations can beused to effect the growth of hair, as for example in the treatment ofalopecia whereby hair growth is potentiated, or for example in cosemeticremoval of hair (depilation) whereby hair growth is inhibited.

In certain embodiments, the subject compositions can be used to inhibit,rather than promote, growth of epithelial-derived tissue. For instance,certain of the compositions disclosed herein may be applied to thetreatment or prevention of a variety hyperplastic or neoplasticconditions. The method can find application for the treatment orprophylaxis of, e.g., psoriasis; keratosis; acne; comedogenic lesions;folliculitis and pseudofolliculitis; keratoacanthoma; callosities;Darier's disease; ichthyosis; lichen planus; molluscous contagiosum;melasma; Fordyce disease; and keloids or hypertrophic scars. Certain ofthe formulations of the present invention may also be used as part oftreatment regimens in auto-immune diseases for affecting healing ofproliferative manifestations of the disorder, as for example, part of atreatment for aphthous ulcers, pemphigus such as pemphigus vulgaris,pemphigus foliaceus, pemphigus vegetans or pemphigus erythematous,epidermolysis, lupus lesions or desquamative lesions.

The subject hedgehog treatments are effective on both human and animalsubjects afflicted with these conditions. Animal subjects to which theinvention is applicable extend to both domestic animals and livestock,raised either as pets or for commercial purposes. Examples are dogs,cats, cattle, horses, sheep, hogs and goats.

Still another aspect of the present invention provides a method ofstimulating the growth and regulating the differentiation of epithelialtissue in tissue culture.

Without wishing to be bound by any particular theory, the induction ofstem cell proliferation by hedgehog proteins may be due at least in partto the ability of these proteins to antagonize (directly or indirectly)patched-mediated regulation of gene expression and other physiologicaleffects mediated by that protein. The patched gene product, a cellsurface protein, is understood to signal through a pathway which causestranscriptional repression of members of the Wnt and Dpp/BMP families ofmorphogens, proteins which impart positional information. In developmentof the CNS and patterning of limbs in vertebrates, the introduction ofhedgehog relieves (derepresses) this inhibition conferred by patched,allowing expression of particular gene programs.

Recently, it has been reported that mutations in the human version ofpatched, a gene first identified in a fruit fly developmental pathway,cause a hereditary skin cancer and may contribute to sporadic skincancers. See, for example, Hahn et al. (1996) Cell 86:841-851; andJohnson et al. (1996) Science 272:1668-1671. The demonstraction thatnevoid basal-cell carcinoma (NBCC) results from mutations in the humanpatched gene provided an example of the roles patched plays inpost-embryonic deveolpment. These observations have led the art tounderstand one activity of patched to be a tumor suppressor gene, whichmay act by inhibiting proliferative signals from hedgehog. Ourobservations set forth below reveal potential new roles for thehedgehog/patched pathway in maintenance of epithelial cell proliferationand differentiation. Accordingly, the present invention contemplates theuse of other agents which are capable of mimicking the effect of thehedgehog protein on patched signalling, e.g., as may be identified fromthe drug screening assays described below.

II. Definitions

For convience, certain terms employed in the specfication, examples, andappended claims are collected here.

The term “hedgehog therapeutic” refers to various forms of hedgehogpolypeptides, as well as peptidomimetics, which can modulate theproliferation/differentiation state of epithelial cells by, as will beclear from the context of individual examples, mimicing or potentiating(agonizing) or inhibiting (antagonizing) the effects of anaturally-occurring hedgehog protein. A hedgehog therapeutic whichmimics or potentiates the activity of a wild-type hedgehog protein is a“hedgehog agonist”. Conversely, a hedgehog therapeutic which inhibitsthe activity of a wild-type hedgehog protein is a “hedgehog antagonist”.

In particular, the term “hedgehog polypeptide” encompasses preparationsof hedgehog proteins and peptidyl fragments thereof, both agonist andantagonist forms as the specific context will make clear.

As used herein the term “bioactive fragment of a hedgehog protein”refers to a fragment of a full-length hedgehog polypeptide, wherein thefragment specifically agonizes or antagonizes inductive events mediatedby wild-type hedgehog proteins. The hedgehog biactive fragmentpreferably is a soluble extracellular portion of a hedgehog protein,where solubility is with reference to physiologically compatiblesolutions. Exemplary bioactive fragments are described in PCTpublications WO 95/18856 and WO 96/17924.

The term “patched” or “ptc” refers to a family of related transmembraneproteins which have been implicated in the signal transduction inducedby contacting a cell with a hedgehog protein. For example, the mammalianptc family includes ptc1 and ptc2. In addition to references set outbelow, see also Takabatake et al. (1997) FEBS Lett 410:485 and GenBankAB000847 for examples of ptc2. Unless otherwise evident from thecontext, it will be understood that embodiments described in the contextof ptc1 (or just ptc) also refer to equivalent embodiments involvingother ptc homologs like ptc2.

The term “ptc therapeutic” refers to agents which either (i) mimic theeffect of hedgehog proteins on patched signalling, e.g., whichantagonize the cell-cycle inhibitory activity of patched, or (ii)activate or potentiate patched signalling. In other embodiments, the ptctherapeutic can be a hedgehog antagonist. The ptc therapeutic can be,e.g., a peptide, a nucleic acid, a carbohydrate, a small organicmolecule, or natural product extract (or fraction thereof).

A “proliferative” form of a hedgehog or ptc therapeutic is one whichinduces proliferation of epithelial cells, particularly epithelial stemcells. Conversely, an “antiproliferative” form of a hedgehog or ptctherapeutic is one which inhibits proliferation of an epithelial cells,preferably in a non-toxic manner, e.g., by promoting or maintaining adifferentiated phenotype or otherwise promoting quiescence.

By way of example, though not wishing to be bound by a particulartheory, proliferative hedgehog polypeptide will generally be a form ofthe protein which derepresses patched-mediated cell-cycle arrest, e.g.,the polypeptide mimics the effect of a naturally occurring hedgehogprotein effect on epithelial cells. A proliferative ptc therapeuticincludes other agents which depress patched-mediated cell-cycle arrest,and may act extracellularly or intracellularly.

An illustrative antiproliferative ptc therapeutic agent may potentiatepatched-mediated cell-cycle arrest. Such agents can be small moleculesthat inhibit, e.g., hedgehog binding to patched, as well as agents whichstimulate and/or potentiate a signal transduction pathway of the patchedprotein.

The terms “epithelia”, “epithelial” and “epithelium” refer to thecellular covering of internal and external body surfaces (cutaneous,mucous and serous), including the glands and other structures derivedtherefrom, e.g., corneal, esophegeal, epidermal, and hair follicleepithelial cells. Other exemplary epithelial tissue includes: olfactoryepithelium, which is the pseudostratified epithelium lining theolfactory region of the nasal cavity, and containing the receptors forthe sense of smell; glandular epithelium, which refers to epitheliumcomposed of secreting cells; squamous epithelium, which refers toepithelium composed of flattened plate-like cells. The term epitheliumcan also refer to transitional epithelium, which that characteristicallyfound lining hollow organs that are subject to great mechanical changedue to contraction and distention, e.g. tissue which represents atransition between stratified squamous and columnar epithelium.

The term “epithelialization” refers to healing by the growth ofepithelial tissue over a denuded surface.

The term “skin” refers to the outer protective covering of the body,consisting of the corium and the epidermis, and is understood to includesweat and sebaceous glands, as well as hair follicle structures.Throughout the present application, the adjective “cutaneous” may beused, and should be understood to refer generally to attributes of theskin, as appropriate to the context in which they are used.

The term “epidermis” refers to the outermost and nonvascular layer ofthe skin, derived from the embryonic ectoderm, varying in thickness from0.07-1.4 mm. On the palmar and plantar surfaces it comprises, fromwithin outward, five layers: basal layer composed of columnar cellsarranged perpendicularly; prickle-cell or spinous layer composed offlattened polyhedral cells with short processes or spines; granularlayer composed of flattened granular cells; clear layer composed ofseveral layers of clear, transparent cells in which the nuclei areindistinct or absent; and horny layer composed of flattened, cornifiednon-nucleated cells. In the epidermis of the general body surface, theclear layer is usually absent.

The “corium” or “dermis” refers to the layer of the skin deep to theepidermis, consisting of a dense bed of vascular connective tissue, andcontaining the nerves and terminal organs of sensation. The hair roots,and sebaceous and sweat glands are structures of the epidermis which aredeeply embedded in the dermis.

The term “nail” refers to the horny cutaneous plate on the dorsalsurface of the distal end of a finger or toe.

The term “epidermal gland” refers to an aggregation of cells associatedwith the epidermis and specialized to secrete or excrete materials notrelated to their ordinary metabolic needs. For example, “sebaceousglands” are holocrine glands in the corium that secrete an oilysubstance and sebum. The term “sweat glands” refers to glands thatsecrete sweat, situated in the corium or subcutaneous tissue, opening bya duct on the body surface.

The term “hair” refers to a threadlike structure, especially thespecialized epidermal structure composed of keratin and developing froma papilla sunk in the corium, produced only by mammals andcharacteristic of that group of animals. Also, the aggregate of suchhairs. A “hair follicle” refers to one of the tubular-invaginations ofthe epidermis enclosing the hairs, and from which the hairs grow; and“hair follicle epithelial cells” refers to epithelial cells whichsurround the dermal papilla in the hair follicle, e.g., stem cells,outer root sheath cells, matrix cells, and inner root sheath cells. Suchcells may be normal non-malignant cells, or transformed/immortalizedcells.

The term “nasal epithelial tissue” refers to nasal and olfactoryepithelium.

“Excisional wounds” include tears, abrasions, cuts, punctures orlacerations in the epithelial layer of the skin and may extend into thedermal layer and even into subcutaneous fat and beyond. Excisionalwounds can result from surgical procedures or from accidentalpenetration of the skin.

“Burn wounds” refer to cases where large surface areas of skin have beenremoved or lost from an individual due to heat and/or chemical agents.

“Dermal skin ulcers” refer to lesions on the skin caused by superficialloss of tissue, usually with inflammation. Dermal skin ulcers which canbe treated by the method of the present invention include decubitusulcers, diabetic ulcers, venous stasis ulcers and arterial ulcers.Decubitus wounds refer to chronic ulcers that result from pressureapplied to areas of the skin for extended periods of time. Wounds ofthis type are often called bedsores or pressure sores. Venous stasisulcers result from the stagnation of blood or other fluids fromdefective veins. Arterial ulcers refer to necrotic skin in the areaaround arteries having poor blood flow.

“Dental tissue” refers to tissue in the mouth which is similar toepithelial tissue, for example gum tissue. The method of the presentinvention is useful for treating periodontal disease.

“Internal epithelial tissue” refers to tissue inside the body which hascharacteristics similar to the epidermal layer in the skin. Examplesinclude the lining of the intestine. The method of the present inventionis useful for promoting the healing of certain internal wounds, forexample wounds resulting from surgery.

A “wound to eye tissue” refers to severe dry eye syndrome, cornealulcers and abrasions and ophthalmic surgical wounds.

Throughout this application, the term “proliferative skin disorder”refers to any disease/disorder of the skin marked by unwanted oraberrant proliferation of cutaneous tissue. These conditions aretypically characterized by epidermal cell proliferation or incompletecell differentiation, and include, for example, X-linked ichthyosis,psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytichyperkeratosis, and seborrheic dermatitis. For example,epidermodysplasia is a form of faulty development of the epidermis.Another example is “epidermolysis”, which refers to a loosened state ofthe epidermis with formation of blebs and bullae either spontaneously orat the site of trauma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate surrounding tissues and to giverise to metastases. Exemplary carcinomas include: “basal cellcarcinoma”, which is an epithelial tumor of the skin that, while seldommetastasizing, has potentialities for local invasion and destruction,“squamous cell carcinoma”, which refers to carcinomas arising fromsquamous epithelium and having cuboid cells; “carcinosarcoma”, whichinclude malignant tumors composed of carcinomatous and sarcomatoustissues; “adenocystic carcinoma”, carcinoma marked by cylinders or bandsof hyaline or mucinous stroma separated or surrounded by nests or cordsof small epithelial cells, occurring in the mammary and salivary glands,and mucous glands of the respiratory tract; “epidermoid carcinoma”,which refers to cancerous cells which tend to differentiate in the sameway as those of the epidermis; i.e., they tend to form prickle cells andundergo cornification; “nasopharyngeal carcinoma”, which refers to amalignant tumor arising in the epithelial lining of the space behind thenose; and “renal cell carcinoma”, which pertains to carcinoma of therenal parenchyma composed of tubular cells in varying arrangements.Another carcinomatous epithelial growth is “papillomas”, which refers tobenign tumors derived from epithelium and having a papillomavirus as acausative agent; and “epidermoidomas”, which refers to a cerebral ormeningeal tumor formed by inclusion of ectodermal elements at the timeof closure of the neural groove.

As used herein, the term “psoriasis” refers to a hyperproliferative skindisorder which alters the skin's regulatory mechanisms. In particular,lesions are formed which involve primary and secondary alterations inepidermal proliferation, inflammatory responses of the skin, and anexpression of regulatory molecules such as lymphokines and inflammatoryfactors. Psoriatic skin is morphologically characterized by an increasedturnover of epidermal cells, thickened epidermis, abnormalkeratinization, inflammatory cell infiltrates into the dermis layer andpolymorphonuclear leukocyte infiltration into the epidermis layerresulting in an increase in the basal cell cycle. Additionally,hyperkeratotic and parakeratotic cells are present.

The term “keratosis” refers to proliferative skin disorder characterizedby hyperplasia of the horny layer of the epidermis. Exemplary keratoticdisorders include keratosis follicularis, keratosis palmaris etplantaris, keratosis pharyngea, keratosis pilaris, and actinickeratosis.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

As used herein, “transformed cells” refers to cells which havespontaneously converted to a state of unrestrained growth, i.e. theyhave acquired the ability to grow through an indefinite number ofdivisions in culture. Transformed cells may be characterized by suchterms as neoplastic, anaplastic and/or hyperplastic, with respect totheir loss of growth control.

As used herein, “immortalized cells” refers to cells which have beenaltered via chemical and/or recombinant means such that the cells havethe ability to grow through an indefinite number of divisions inculture.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human animal.

The term “cosmetic preparation” refers to a form of a pharmaceuticalpreparation which is formulated for topical administration.

An “effective amount” of, e.g., a hedgehog therapeutic, with respect tothe subject method of treatment, refers to an amount of, e.g., ahedgehog polypeptide in a preparation which, when applied as part of adesired dosage regimen brings about a change in the rate of cellproliferation and/or the state of differentiation of a cell so as toproduce an amount of epithelial cell proliferation according toclinically acceptable standards for the disorder to be treated or thecosmetic purpose.

The “growth state” of a cell refers to the rate of proliferation of thecell and the state of differentiation of the cell.

“Homology” and “identity” each refer to sequence similarity between twopolypeptide sequences, with identity being a more strict comparison.Homology and identity can each be determined by comparing a position ineach sequence which may be aligned for purposes of comparison. When aposition in the compared sequence is occupied by the same amino acidresidue, then the polypeptides can be referred to as identical at thatposition; when the equivalent site is occupied by the same amino acid(e.g., identical) or a similar amino acid (e.g., similar in stericand/or electronic nature), then the molecules can be refered to ashomologous at that position. A percentage of homology or identitybetween sequences is a function of the number of matching or homologouspositions shared by the sequences. An “unrelated” or “non-homologous”sequence shares less than 40 percent identity, though preferably lessthan 25 percent identity, with an AR sequence of the present invention.

The term “corresponds to”, when referring to a particular polypeptide ornucleic acid sequence is meant to indicate that the sequence of interestis identical or homologous to the reference sequence to which it is saidto correspond.

The terms “recombinant protein”, “heterologous protein” and “exogenousprotein” are used interchangeably throughout the specification and referto a polypeptide which is produced by recombinant DNA techniques,wherein generally, DNA encoding the polypeptide is inserted into asuitable expression construct which is in turn used to transform a hostcell to produce the heterologous protein. That is, the polypeptide isexpressed from a heterologous nucleic acid.

A “chimeric protein” or “fusion protein” is a fusion of a first aminoacid sequence encoding a hedgehog polypeptide with a second amino acidsequence defining a domain foreign to and not substantially homologouswith any domain of hh protein. A chimeric protein may present a foreigndomain which is found (albeit in a different protein) in an organismwhich also expresses the first protein, or it may be an “interspecies”,“intergenic”, etc. fusion of protein structures expressed by differentkinds of organisms. In general, a fusion protein can be represented bythe general formula (X)_(n)-(hh)_(m)-(Y)_(n), wherein hh represents allor a portion of the hedgehog protein. X and Y each independentlyrepresent an amino acid sequences which are not naturally found as apolypeptide chain contiguous with the hedgehog sequence, m is an integergreater than or equal to 1, and each occurrence of n is, independently,0 or an integer greater than or equal to 1 (n and m are preferably nogreater than 5 or 10).

III. Exemplary Applications of Method and Compositions

The subject method has wide applicability to the treatment orprophylaxis of disorders afflicting epithelial tissue, as well as incosmetic uses. In general, the method can be characterized as includinga step of administering to an animal an amount of a ptc or hedgehogtherapeutic effective to alter the proliferative state of a treatedepithelial tissue. The mode of administration and dosage regimens willvary depending on the epithelial tissue(s) which is to be treated. Forexample, topical formulations will be preferred where the treated tissueis epidermal tissue, such as dermal or mucosal tissues. Likewise, asdescribed in further detail below, the use of a particular ptc orhedgehog therapeutic, e.g., an agonist or antagonist, will depend onwhether proliferation of cells of the treated tissue is desired orintended to be prevented.

A method which “promotes the healing of a wound” results in the woundhealing more quickly as a result of the treatment than a similar woundheals in the absence of the treatment. “Promotion of wound healing” canalso mean that the method causes the proliferation and growth of, interalia, keratinocytes, or that the wound heals with less scarring, lesswound contraction, less collagen deposition and more superficial surfacearea. In certain instances, “promotion of wound healing” can also meanthat certain methods of wound healing have improved success rates, (e.g.the take rates of skin grafts,) when used together with the method ofthe present invention.

Complications are a constant risk with wounds that have not fully healedand remain open. Although most wounds heal quickly without treatment,some types of wounds resist healing. Wounds which cover large surfaceareas also remain open for extended periods of time. In one embodimentof the present invention, the subject method can be used to acceleratethe healing of wounds involving epithelial tissues, such as resultingfrom surgery, bums, inflammation or irritation. Certain of the hedgehogand ptc therapeutic formulations (e.g., proliferative forms) of thepresent invention can also be applied prophylactically, such as in theform of a cosmetic preparation, to enhance tissue regenerationprocesses, e.g., of the skin, hair and/or fingernails.

Despite significant progress in reconstructive surgical techniques,scarring can be an important obstacle in regaining normal function andappearance of healed skin. This is particularly true when pathologicscarring such as keloids or hypertrophic scars of the hands or facecauses functional disability or physical deformity. In the severestcircumstances, such scarring may precipitate psychosocial distress and alife of economic deprivation. Wound repair includes the stages ofhemostasis, inflammation, proliferation, and remodeling. Theproliferative stage involves multiplication of fibroblasts andendothelial and epithelial cells. Through the use of the subject method,the rate of proliferation of epithelial cells in and proximal to thewound can be controlled in order to accelerate closure of the woundand/or minimize the formation of scar tissue.

Full and partial thickness bums are an example of a wound type whichoften covers large surface areas and therefore requires prolongedperiods of time to heal. As a result, life-threatening complicationssuch as infection and loss of bodily fluids often arise. In addition,healing in burns is often disorderly, resulting in scarring anddisfigurement. In some cases wound contraction due to excessive collagendeposition results in reduced mobility of muscles in the vicinity of thewound. The compositions and method of the present invention can be usedto accelerate the rate of healing of burns and to promote healingprocesses that result in more desirable cosmetic outcomes and less woundcontraction and scarring.

Severe burns which cover large areas are often treated by skinautografts taken from undamaged areas of the patient's body. The subjectmethod can also be used in conjunction with skin grafts to impove “take”rates of the graft by accelerating growth of both the grafted skin andthe patient's skin that is proximal to the graft.

Dermal ulcers are yet another example of wounds that are amenable totreatment by the subject method, e.g., to cause healing of the ulcerand/or to prevent the ulcer from becoming a chronic wound. For example,one in seven individuals with diabetes develop dermal ulcers on theirextremities, which are susceptible to infection. Individuals withinfected diabetic ulcers often require hospitalization, intensiveservices, expensive antibiotics, and, in some cases, amputation. Dermalulcers, such as those resulting from venous disease (venous stasisulcers), excessive pressure (decubitus ulcers) and arterial ulcers alsoresist healing. The prior art treatments are generally limited tokeeping the wound protected, free of infection and, in some cases, torestore blood flow by vascular surgery. According to the present method,the afflicted area of skin can be treated by a therapy which includes ahedgehog or ptc therapeutic which promotes epithelization of the wound,e.g., accelerates the rate of the healing of the skin ulcers.

The present treatment can also be effective as part of a therapeuticregimen for treating oral and paraoral ulcers, e.g. resulting fromradiation and/or chemotherapy. Such ulcers commonly develop within daysafter chemotherapy or radiation therapy. These ulcers usually begin assmall, painful irregularly shaped lesions usually covered by a delicategray necrotic membrane and surrounded by inflammatory tissue. In manyinstances, lack of treatment results in proliferation of tissue aroundthe periphery of the lesion on an inflammatory basis. For instance, theepithelium bordering the ulcer usually demonstrates proliferativeactivity, resulting in loss of continuity of surface epithelium. Theselesions, because of their size and loss of epithelial integrity, lendthe body to potential secondary infection. Routine ingestion of food andwater becomes a very painful event and, if the ulcers proliferatethroughout the alimentary canal, diarrhea usually is evident with allits complicating factors. According to the present invention, atreatment for such ulcers which includes application of an hedgehogtherapeutic can reduce the abnormal proliferation and differentiation ofthe affected epithelium, helping to reduce the severity of subsequentinflammatory events.

In another exemplary embodiment, the subject method is provided fortreating or preventing gastrointestinal diseases. Briefly, a widevariety of diseases are associated with disruption of thegastrointestinal epithelium or villi, including chemotherapy- andradiation-therapy-induced enteritis (i.e. gut toxicity) and mucositis,peptic ulcer disease, gastroenteritis and colitis, villus atrophicdisorders, and the like. For example, chemotherapeutic agents andradiation therapy used in bone marrow transplantation and cancer therapyaffect rapidly proliferating cells in both the hematopoietic tissues andsmall intestine, leading to severe and often dose-limiting toxicities.Damage to the small intestine mucosal barrier results in seriouscomplications of bleeding and sepsis. The subject method can be used topromote proliferation of gastrointenstinal epithelium and therebyincrease the tolerated doses for radiation and chemotherapy agents.Effective treatment of gastrointestinal diseases may be determined byseveral criteria, including an enteritis score, other tests well knownin the art.

The subject method and compositions can also be used to treat woundsresulting from dermatological diseases, such as lesions resulting fromautoimmune disorders such as psoriasis. Atopic dermititis refers to skintrauma resulting from allergies associated with an immune responsecaused by allergens such as pollens, foods, dander, insect venoms andplant toxins.

With age, the epidermis thins and the skin appendages atrophy. Hairbecomes sparse and sebaceous secretions decrease, with consequentsusceptibility to dryness, chapping, and fissuring. The dermisdiminishes with loss of elastic and collagen fibers. Moreover,keratinocyte proliferation (which is indicative of skin thickness andskin proliferative capacity) decreases with age. An increase inkeratinocyte proliferation is believed to conteract skin aging, i.e.,wrinkles, thickness, elasticity and repair. According to the presentinvention, a proliferative form of a hedgehog or ptc therapeutic can beused either therapeutically or cosmetically to counteract, at least fora time, the effects of aging on skin.

The subject method can also be used in treatment of a wound to eyetissue. Generally, damage to corneal tissue, whether by disease, surgeryor injury, may affect epithelial and/or endothelial cells, depending onthe nature of the wound. Corneal epithelial cells are thenon-keratinized epithelial cells lining the external surface of thecornea and provide a protective barrier against the externalenvironment. Corneal wound healing has been of concern to bothclinicians and researchers. Opthomologists are frequently confrontedwith corneal dystrophies and problematic injuries that result inpersistent and recurrent epithelial erosion, often leading to permanentendothelial loss. The use of proliferative forms of the subject hedgehogand/or other ptc therapeutics can be used in these instances to promoteepithelialization of the affected corneal tissue.

To further illustrate, specific disorders typically associated withepithelial cell damage in the eye, and for which the subject method canprovide beneficial treatment, include persistent corneal epithelialdefects, recurrent erosions, neurotrophic corneal ulcers,keratoconjunctivitis sicca, microbial corneal ulcers, viral corneaulcers, and the like. Surgical procedures typically causing injury tothe epithelial cell layers include laser procedures performed on theocular surface, any refractive surgical procedures such as radialkeratotomy and astigmatic keratotomy, conjunctival flaps, conjunctivaltransplants, epikeratoplasty, and corneal scraping. Moreover,superficial wounds such as scrapes, surface erosion, inflammation, etc.can cause lose of epithelial cells. According to the present invention,the corneal epithelium is contacted with an amount of a ptc or hedgehogtherapeutic effective to cause proliferation of the corneal epithelialcells to appropriately heal the wound.

In other embodiments, antiproliferative preparations of hedgehog or ptctherapeutics can be used to inhibit lens epithelial cell proliferationto prevent post-operative complications of extracapsular cataractextraction. Cataract is an intractable eye disease and various studieson a treatment of cataract have been made. But at present, the treatmentof cataract is attained by surgical operations. Cataract surgery hasbeen applied for a long time and various operative methods have beenexamined. Extracapsular lens extraction has become the method of choicefor removing cataracts. The major medical advantages of this techniqueover intracapsular extraction are lower incidence of aphakic cystoidmacular edema and retinal detachment. Extracapsular extraction is alsorequired for implantation of posterior chamber type intraocular lenseswhich are now considered to be the lenses of choice in most cases.

However, a disadvantage of extracapsular cataract extraction is the highincidence of posterior lens capsule opacification, often calledafter-cataract, which can occur in up to 50% of cases within three yearsafter surgery. After-cataract is caused by proliferation of equatorialand anterior capsule lens epithelial cells which remain afterextracapsular lens extraction. These cells proliferate to causeSommerling rings, and along with fibroblasts which also deposit andoccur on the posterior capsule, cause opacification of the posteriorcapsule, which interferes with vision. Prevention of after-cataractwould be preferable to treatment. To inhibit secondary cataractformation, the subject method provides a means for inhibitingproliferation of the remaining lens epithelial cells. For example, suchcells can be induced to remain quiescent by instilling a solutioncontaining an antiproliferative hedgehog or ptc therapeutic preparationinto the anterior chamber of the eye after lens removal. Furthermore,the solution can be osmotically balanced to provide minimal effectivedosage when instilled into the anterior chamber of the eye, therebyinhibiting subcapsular epithelial growth with some specificity.

The subject method can also be used in the treatment of corneopathiesmarked by corneal epithelial cell proliferation, as for example inocular epithelial disorders such as epithelial downgrowth or squamouscell carcinomas of the ocular surface.

The maintenance of tissues and organs ex vivo is also highly desirable.Tissue replacement therapy is well established in the treatment of humandisease. For example, more than 40,000 corneal transplants wereperformed in the United States in 1996. Human epidermal cells can begrown in vitro and used to populate burn sites and chronic skin ulcersand other dermal wounds. The subject method can be used to acceleratethe growth of epithelial tissue in vitro, as well as to accelerate thegrafting of the cultured epithelial tissue to an animal host

The present method can be used for improving the “take rate” of a skingraft. Grafts of epidermal tissue can, if the take rate of the graft isto long, blister and shear, decreasing the likelihood that the autograftwill “take”, i.e. adhere to the wound and form a basement membrane withthe underlying granulation tissue. Take rates can be increased by thesubject method by inducing proliferation of the keratinocytes. Themethod of increasing take rates comprises contacting the skin autograftwith an effective wound healing amount of a hedgehog or ptc therapeuticcompositions described in the method of promoting wound healing and inthe method of promoting the growth and proliferation of keratinocytes,as described above.

Skin equivalents have many uses not only as a replacement for human oranimal skin for skin grafting, but also as test skin for determining theeffects of pharmaceutical substances and cosmetics on skin. A majordifficulty in pharmacological, chemical and cosmetic testing is thedifficulties in determining the efficacy and safety of the products onskin. One advantage of the skin equivalents of the invention is theiruse as an indicator of the effects produced by such substances throughin vitro testing on test skin.

Thus, in one embodiment of the subject method can be used as part of aprotocol for skin grafting of, e.g., denuded areas, granulating woundsand burns. The use of proliferative hedgehog and/or ptc therapeutics canenhance such grafting techniques as split thickness autografts andepidermal autografts (cultured autogenic keratinocytes) and epidermalallografts (cultured allogenic keratinocytes). In the instance of theallograft, the use of the subject method to enhance the formation ofskin equivalents in culture helps to provide/maintain a ready supply ofsuch grafts (e.g., in tissue banks) so that the patients might becovered in a single procedure with a material which allows permanenthealing to occur.

In this regard, the present invention also concerns composite livingskin equivalents comprising an epidermal layer of cultured keratinocytecells which have been expanded by treatment with a hedgehog or other ptctherapeutic. The subject method can be used as part of a process for thepreparation of composite living skin equivalents. In an illustrativeembodiment, such a method comprises obtaining a skin sample, treatingthe skin sample enzymically to separate the epidermis from the dermis,treating the epidermis enzymically to release the keratinocyte cells,culturing, in the presence of a hedgehog or ptc therapeutic, theepidermal keratinocytes until confluence, in parallel, or separately,treating the dermis enzymatically to release the fibroblast cells,culturing the fibroblasts cells until sub-confluence, inoculating aporous, cross-linked collagen sponge membrane with the culturedfibroblast cells, incubating the inoculated collagen sponge on itssurface to allow the growth of the fibroblast cells throughout thecollagen sponge, and then inoculating it with cultured keratinocytecells, and further incubating the composite skin equivalent complex inthe presence of a hedgehog or ptc therapeutic to promote the growth ofthe cells.

In other embodiments, skin sheets containing both epithelial andmesenchymal layers can be isolated in culture and expanded with culturemedia supplemented with a proliferative form of a hedgehog or ptctherapeutic.

Any skin sample amenable to cell culture techniques can be used inaccordance with the present invention. The skin samples may be autogenicor allogenic.

In another aspect of the invention, the subject method can be used inconjunction with various periodontal procedures in which control ofepithelial cell proliferation in and around periodontal tissue isdesired.

In one embodiment, proliferative forms of the hedgehog and ptctherapeutics can be used to enhance reepithelialization around naturaland prosthetic teeth, e.g., to promote formation of gum tissue.

In another embodiment, antiproliferative ptc therapeutics can findapplication in the treatment of peridontal disease. It is estimated thatin the United States alone, there are in excess of 125 million adultswith periodontal disease in varying forms. Periodontal disease starts asinflammatory lesions because of specific bacteria localizing in the areawhere the gingiva attaches to the tooth. Usually first to occur is avascular change in the underlying connective tissue. Inflammation in theconnective tissue stimulates the following changes in the epitheliallining of the sulcus and in the epithelial attachment: increased mitoticactivity in the basal epithelial layer; increased producing of keratinwith desquamation; cellular desquamation adjacent to the tooth surfacetends to deepen the pocket; epithelial cells of the basal layer at thebottom of the sulcus and in the area of attachment proliferate into theconnective tissue and break up of the gingival fibers begins to occur,wherein dissolution of the connective tissue results in the formation ofan open lesion. The application of hedgehog preparations to theperiodontium can be used to inhibit proliferation of epithelial tissueand thus prevent further periodontoclastic development.

In yet another aspect, the subject method can be used to help controlguided tissue regeneration, such as when used in conjunction withbioresorptable materials. For example, incorporation of periodontalimplants, such as prosthetic teeth, can be facilitated by the instantmethod. Reattachment of a tooth involves both formation of connectivetissue fibers and re-epithelization of the tooth pocket. The subjectmethodtreatment can be used to accelerate tissue reattachment bycontrolling the mitotic function of basal epithelial cells in earlystages of wound healing.

Yet another aspect of the present invention relates to the use ofhedgehog therapeutic preparations to control hair growth. Hair isbasically composed of keratin, a tough and insoluble protein; its chiefstrength lies in its disulphide bond of cystine. Each individual haircomprises a cylindrical shaft and a root, and is contained in afollicle, a flask-like depression in the skin. The bottom of thefollicle contains a finger-like projection termed the papilla, whichconsists of connective tissue from which hair grows, and through whichblood vessels supply the cells with nourishment. The shaft is the partthat extends outwards from the skin surface, whilst the root has beendescribed as the buried part of the hair. The base of the root expandsinto the hair bulb, which rests upon the papilla. Cells from which thehair is produced grow in the bulb of the follicle; they are extruded inthe form of fibers as the cells proliferate in the follicle. Hair“growth” refers to the formation and elongation of the hair fiber by thedividing cells.

As is well known in the art, the common hair cycle is divided into threestages: anagen, catagen and telogen. During the active phase (anagen),the epidermal stem cells of the dermal papilla divide rapidly. Daughtercells move upward and differentiate to form the concentric layers of thehair itself. The transitional stage, catagen, is marked by the cessationof mitosis of the stem cells in the follicle. The resting stage is knownas telogen, where the hair is retained within the scalp for severalweeks before an emerging new hair developing below it dislodges thetelogen-phase shaft from its follicle. From this model it has becomeclear that the larger the pool of dividing stem cells that differentiateinto hair cells, the more hair growth occurs. Accordingly, methods forincreasing or reducing hair growth can be carried out by potentiating orinhibiting, respectively, the proliferation of these stem cells.

In one embodiment, the subject method provides a means for altering thedynamics of the hair growth cycle to induce proliferation of hairfollicle cells, particularly stem cells of the hair follicle. Thesubject compositions and method can be used to increase hair folliclesize and the rate of hair growth in warm-blooded animals, such ashumans, e.g., by promoting proliferation of hair follicle stem cells. Inone embodiment, the method comprises administering to the skin in thearea in which hair growth is desired an amount of hedgehog or ptctherapeutic sufficient to increase hair follicle size and/or the rate ofhair growth in the animal. Typically, the composition will beadministered topically as a cream, and will be applied on a daily basisuntil hair growth is observed and for a time thereafter sufficient tomaintain the desired amount of hair growth. This method can haveapplications in the promotion of new hair growth or stimulation of therate of hair growth, e.g., following chemotherapeutic treatment or fortreating various forms of alopecia, e.g., male pattern baldness. Forinstance, one of several biochemical cellular and molecular disturbancesthat occur during the anagen phase or catagen phase of subjects withandrogenic alopecia can be corrected or improved by treatment using thesubject method, e.g., in the functioning or formation of the stem cells,their migration process or during the mitosis phase of keratinproduction within the follicular papilla and matrix.

In other embodimemts, cerain of the hedgehog and ptc therapeutics (e.g.,antiproliferative forms) can be employed as a way of reducing the growthof human hair as opposed to its conventional removal by cutting,shaving, or depilation. For instance, the present method can be used inthe treatment of trichosis characterized by abnormally rapid or densegrowth of hair, e.g. hypertrichosis. In an exemplary embodiment,hedgehog antagonists can be used to manage hirsutism, a disorder markedby abnormal hairiness. The subject method can also provide a process forextending the duration of depilation.

Moreover, because a hedgehog antagonist (or ptc agonist) will often becytostatic to epithelial cells, rather than cytotoxic, such agents canbe used to protect hair follicle cells from cytotoxic agents whichrequire progression into S-phase of the cell-cycle for efficacy, e.g.radiation-induced death. Treatment by the subject method can provideprotection by causing the hair follicle cells to become quiescent, e.g.,by inhibiting the cells from entering S phase, and thereby preventingthe follicle cells from undergoing mitotic catastrophe or programmedcell death. For instance, hedgehog antagonists can be used for patientsundergoing chemo- or radiation-therapies which ordinarily result in hairloss. By inhibiting cell-cycle progression during such therapies, thesubject treatment can protect hair follicle cells from death which mightotherwise result from activation of cell death programs. After thetherapy has concluded, the hedgehog or ptc treatment can also be removedwith concommitant relief of the inhibition of follicle cellproliferation.

The subject method can also be used in the treatment of folliculitis,such as folliculitis decalvans, folliculitis ulerythematosa reticulataor keloid folliculitis. For example, a cosmetic prepration of anhedgehog therapeutic can be applied topically in the treatment ofpseudofolliculitis, a chronic disorder occurring most often in thesubmandibular region of the neck and associated with shaving, thecharacteristic lesions of which are erythematous papules and pustulescontaining buried hairs.

In another aspect of the invention, antiproliferative forms of thesubject hedgehog and ptc therapeutics can be used to inducedifferentiation of epithelially-derived tissue. Such forms of thesemolecules can provide a basis for differentiation therapy for thetreatment of hyperplastic and/or neoplastic conditions involvingepithelial tissue. For example, such preparations can be used for thetreatment of cutaneous diseases in which there is abnormal proliferationor growth of cells of the skin.

For instance, the pharmaceutical preparations of the invention areintended for the treatment of hyperplastic epidermal conditions, such askeratosis, as well as for the treatment of neoplastic epidermalconditions such as those characterized by a high proliferation rate forvarious skin cancers, as for example basal cell carcinoma or squamouscell carcinoma. The subject method can also be used in the treatment ofautoimmune diseases affecting the skin, in particular, of dermatologicaldiseases involving morbid proliferation and/or keratinization of theepidermis, as for example, caused by psoriasis or atopic dermatosis.

Many common diseases of the skin, such as psoriasis, squamous cellcarcinoma, keratoacanthoma and actinic keratosis are characterized bylocalized abnormal proliferation and growth. For example, in psoriasis,which is characterized by scaly, red, elevated plaques on the skin, thekeratinocytes are known to proliferate much more rapidly than normal andto differentiate less completely.

In one embodiment, the preparations of the present invention aresuitable for the treatment of dermatological ailments linked tokeratinization disorders causing abnormal proliferation of skin cells,which disorders may be marked by either inflammatory or non-inflammatorycomponents. To illustrate, therapeutic preparations of a ptc agonist,e.g., which promotes quiescense or differentiation can be used to treatvarying forms of psoriasis, be they cutaneous, mucosal or ungual.Psoriasis, as described above, is typically characterized by epidermalkeratinocytes which display marked proliferative activation anddifferentiation along a “regenerative” pathway. Treatment with anantiproliferative embodiment of the subject method can be used toreverse the pathological epidermal activiation and can provide a basisfor sustained remission of the disease.

A variety of other keratotic lesions are also candidates for treatmentwith the subject antiproliferative preparations. Actinic keratoses, forexample, are superficial inflammatory premalignant tumors arising onsun-exposed and irradiated skin. The lesions are erythematous to brownwith variable scaling. Current therapies include excisional andcryosurgery. These treatments are painful, however, and often producecosmetically unacceptable scarring. Accordingly, treatment of keratosis,such as actinic keratosis, can include application, preferably topical,of a ptc agonist composition in amounts sufficient to inhibithyperproliferation of epidermal/epidermoid cells of the lesion.

Acne represents yet another dermatologic ailment which may be treatedwith an antiproliferative embodiment of the subject method. Acnevulgaris, for instance, is a multifactorial disease most commonlyoccurring in teenagers and young adults, and is characterized by theappearance of inflammatory and noninflammatory lesions on the face andupper trunk. The basic defect which gives rise to acne vulgaris ishypercornification of the duct of a hyperactive sebaceous gland.Hypercornification blocks the normal mobility of skin and folliclemicroorganisms, and in so doing, stimulates the release of lipases byPropinobacterium acnes and Staphylococcus epidermidis bacteria andPitrosporum ovale, a yeast. Treatment with an antiproliferative form ofa hedgehog or ptc therapeutic, particularly topical preparations, may beuseful for preventing the transitional features of the ducts, e.g.hypercornification, which lead to lesion formation. The subjecttreatment may further include, for example, antibiotics, retinoids andantiandrogens.

The present invention also provides a method for treating various formsof dermatitis. Dermatitis is a descriptive term referring to poorlydemarcated lesions which are either pruritic, erythematous, scaley,blistered, weeping, fissured or crusted. These lesions arise from any ofa wide variety of causes. The most common types of dermatitis areatopic, contact and diaper dermatitis. For instance, seborrheicdermatitis is a chronic, usually pruritic, dermatitis with erythema,dry, moist, or greasy scaling, and yellow crusted patches on variousareas, especially the scalp, with exfoliation of an excessive amount ofdry scales stasis dermatitis, an often chronic, usually eczematousdermatitis. Actinic dermatitis is dermatitis that due to exposure toactinic radiation such as that from the sun, ultraviolet waves or x- orgamma-radiation. According to the present invention, the subjecthedgehog or ptc therapeutic preparations can be used in the treatmentand/or prevention of certain symptoms of dermatitis caused by unwantedproliferation of epithelial cells. Such therapies for these variousforms of dermatitis can also include topical and systemiccorticosteroids, antipuritics, and antibiotics.

Also included in ailments which may be treated by the subject method aredisorders specific to non-humans, such as mange.

IV. Exemplary Hedgehog Therapeutic Compounds

The hedgehog therapeutic compositions of the subject method can begenerated by any of a variety of techniques, including purification ofnaturally occurring proteins, recombinantly produced proteins andsynthetic chemistry. Polypeptide forms of the hedgehog therapeutics arepreferably derived from vertebrate hedgehog proteins, e.g., havesequences corresponding to naturally occurring hedgehog proteins, orfragments thereof, from vertebrate organisms. However, it will beappreciated that the hedgehog polypeptide can correspond to a hedgehogprotein (or fragment thereof) which occurs in any metazoan organism.

The various naturally-occurring hedgehog proteins from which the subjecttherapeutics can be derived are characterized by a signal peptide, ahighly conserved N-terminal region, and a more divergent C-terminaldomain. In addition to signal sequence cleavage in the secretory pathway(Lee, J. J. et al. (1992) Cell 71:33-50; Tabata, T. et al. (1992) GenesDev. 2635-2645; Chang, D. E. et al. (1994) Development 120:3339-3353),hedgehog precursor proteins naturally undergo an internalautoproteolytic cleavage which depends on conserved sequences in theC-terminal portion (Lee et al. (1994) Science 266:1528-1537; Porter etal. (1995) Nature 374:363-366). This autocleavage leads to a 19 kDN-terminal peptide and a C-terminal peptide of 26-28 kD (Lee et al.(1992) supra; Tabata et al. (1992) supra; Chang et al. (1994) supra, Leeet al. (1994) supra; Bumcrot, D. A., et al. (1995) Mol. Cell. Biol.15:2294-2303; Porter et al. (1995) supra; Ekker, S. C. et al. (1995)Curr. Biol. 5:944-955; Lai, C. J. et al. (1995) Development121:2349-2360). The N-terminal peptide stays tightly associated with thesurface of cells in which it was synthesized, while the C-terminalpeptide is freely diffusible both in vitro and in vivo (Lee et al.(1994) supra; Bumcrot et al. (1995) supra; Mart′, E. et al. (1995)Development 121:2537-2547; Roelink, H. et al. (1995) Cell 81:445-455).Cell surface retention of the N-terminal peptide is dependent onautocleavage, as a truncated form of hedgehog encoded by an RNA whichterminates precisely at the normal position of internal cleavage isdiffusible in vitro (Porter et al. (1995) supra) and in vivo (Porter, J.A. et al. (1996) Cell 86, 21-34). Biochemical studies have shown thatthe autoproteolytic cleavage of the hedgehog precursor protein proceedsthrough an internal thioester intermediate which subsequently is cleavedin a nucleophilic substitution. It is suggested that the nucleophile isa small lipophilic molecule, more particularly cholesterol, whichbecomes covalently bound to the C-terminal end of the N-peptide (Porteret al. (1996) supra), tethering it to the cell surface.

The vertebrate family of hedgehog genes includes at least four members,e.g., paralogs of the single drosophila hedgehog gene (SEQ ID No. 19).Three of these members, herein referred to as Desert hedgehog (Dhh),Sonic hedgehog (Shh) and Indian hedgehog (Ihh), apparently exist in allvertebrates, including fish, birds, and mammals. A fourth member, hereinreferred to as tiggie-winkle hedgehog (Thh), appears specific to fish.According to the appended sequence listing, (see also Table 1) a chickenShh polypeptide is encoded by SEQ ID No: 1; a mouse Dhh polypeptide isencoded by SEQ ID No:2; a mouse Ihh polypeptide is encoded by SEQ IDNo:3; a mouse Shh polypeptide is encoded by SEQ ID No:4 a zebrafish Shhpolypeptide is encoded by SEQ ID No:5; a human Shh polypeptide isencoded by SEQ ID No:6; a human Ihh polypeptide is encoded by SEQ IDNo:7; a human Dhh polypeptide is encoded by SEQ ID No.8; and a zebrafishThh is encoded by SEQ ID No. 9.

TABLE 1 Guide to hedgehog sequences in Sequence Listing Nucleotide AminoAcid Chicken Shh SEQ ID No. 1 SEQ ID No. 10 Mouse Dhh SEQ ID No. 2 SEQID No. 11 Mouse Ihh SEQ ID No. 3 SEQ ID No. 12 Mouse Shh SEQ ID No. 4SEQ ID No. 13 Zebrafish Shh SEQ ID No. 5 SEQ ID No. 14 Human Shh SEQ IDNo. 6 SEQ ID No. 15 Human Ihh SEQ ID No. 7 SEQ ID No. 16 Human Dhh SEQID No. 8 SEQ ID No. 17 Zebrafish Thh SEQ ID No. 9 SEQ ID No. 18Drosophila HH SEQ ID No. 19 SEQ ID No. 20

In addition to the sequence variation between the various hedgehoghomologs, the hedgehog proteins are apparently present naturally in anumber of different forms, including a pro-form, a full-length matureform, and several processed fragments thereof. The pro-form includes anN-terminal signal peptide for directed secretion of the extracellulardomain, while the full-length mature form lacks this signal sequence.

As described above, further processing of the mature form occurs in someinstances to yield biologically active fragments of the protein. Forinstance, sonic hedgehog undergoes additional proteolytic processing toyield two peptides of approximately 19 kDa and 27 kDa, the 19 kDafragment corresponding to an proteolytic N-terminal portion of themature protein.

In addition to proteolytic fragmentation, the vertebrate hedgehogproteins can also be modified post-translationally, such as byglycosylation and/or addition of lipophilic moieties, such as stents,fatty acids, etc., though bacterially produced (e.g. unmodified) formsof the proteins still maintain certain of the bioactivities of thenative protein. Bioactive fragments of hedgehog polypeptides of thepresent invention have been generated and are described in great detailin, e.g., PCT publications WO 95/18856 and WO 96/17924.

There are a wide range of lipophilic moieties with which hedgehogpolypeptides can be derivatived. The term “lipophilic group”, in thecontext of being attached to a hedgehog polypeptide, refers to a grouphaving high hydrocarbon content thereby giving the group high affinityto lipid phases. A lipophilic group can be, for example, a relativelylong chain alkyl or cycloalkyl (preferably n-alkyl) group havingapproximately 7 to 30 carbons. The alkyl group may terminate with ahydroxy or primary amine “tail”. To further illustrate, lipophilicmolecules include naturally-occurring and synthetic aromatic andnon-aromatic moieties such as fatty acids, sterols, esters and alcohols,other lipid molecules, cage structures such as adamantane andbuckminsterfullerenes, and aromatic hydrocarbons such as benzene,perylene, phenanthrene, anthracene, naphthalene, pyrene, chrysene, andnaphthacene.

In one embodiment, the hedgehog polypeptide is modified with one or moresterol moieties, such as cholesterol. See, for example, PCT publicationWO 96/17924. In certain embodiments, the cholesterol is preferably addedto the C-terminal glycine were the hedgehog polypeptide corresponds tothe naturally-occurring N-terminal proteolytic fragment.

In another embodiment, the hedgehog polypeptide can be modified with afatty acid moiety, such as a myrostoyl, palmitoyl, stearoyl, orarachidoyl moiety. See, e.g., Pepinsky et al. (1998) J Biol. Chem 273:14037.

In addition to those effects seen by cholesterol-addition to theC-terminus or fatty acid addition to the N-terminus of extracellularfragments of the protein, at least certain of the biological activitiesof the hedgehog gene products are unexpectedly potentiated byderivativation of the protein with lipophilic moieties at other sites onthe protein and/or by moieties other than cholesterol or fatty acids.Certain aspects of the invention are directed to the use of preparationsof hedgehog polypeptides which are modified at sites other thanN-terminal or C-terminal residues of the natural processed form of theprotein, and/or which are modified at such terminal residues withlipophilic moieties other than a sterol at the C-terminus or fatty acidat the N-terminus.

Particularly useful as lipophilic molecules are alicyclic hydrocarbons,saturated and unsaturated fatty acids and other lipid and phospholipidmoieties, waxes, cholesterol, isoprenoids, terpenes and polyalicyclichydrocarbons including adamantane and buckminsterfullerenes, vitamins,polyethylene glycol or oligoethylene glycol, (C1-C18)-alkyl phosphatediesters, —O—CH2—CH(OH)—O—(C12-C18)-alkyl, and in particular conjugateswith pyrene derivatives. The lipophilic moiety can be a lipophilic dyesuitable for use in the invention include, but are not limited to,diphenylhexatriene, Nile Red, N-phenyl-1-naphthylamine, Prodan,Laurodan, Pyrene, Perylene, rhodamine, rhodamine B,tetramethylrhodamine, Texas Red, sulforhodamine,1,1′-didodecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate,octadecyl rhodamine B and the BODIPY dyes available from MolecularProbes Inc.

Other exemplary lipophilic moietites include aliphatic carbonyl radicalgroups include 1- or 2-adamantylacetyl, 3-methyladamant-1-ylacetyl,3-methyl-3-bromo-1-adamantylacetyl, 1-decalinacetyl, camphoracetyl,camphaneacetyl, noradamantylacetyl, norbomaneacetyl,bicyclo[2.2.2.]-oct-5-eneacetyl,1-methoxybicyclo[2.2.2.]-oct-5-ene-2-carbonyl,cis-5-norbornene-endo-2,3-dicarbonyl, 5-norbornen-2-ylacetyl,(1R)-(−)-myrtentaneacetyl, 2-norbornaneacetyl,anti-3-oxo-tricyclo[2.2.1.0<2,6>]-heptane-7-carbonyl, decanoyl,dodecanoyl, dodecenoyl, tetradecadienoyl, decynoyl or dodecynoyl.

The hedgehog polypeptide can be linked to the hydrophobic moiety in anumber of ways including by chemical coupling means, or by geneticengineering.

There are a large number of chemical cross-linking agents that are knownto those skilled in the art. For the present invention, the preferredcross-linking agents are heterobifunctional cross-linkers, which can beused to link the hedgehog polypeptide and hydrophobic moiety in astepwise manner. Heterobifunctional cross-linkers provide the ability todesign more specific coupling methods for conjugating to proteins,thereby reducing the occurrences of unwanted side reactions such ashomo-protein polymers. A wide variety of heterobifunctionalcross-linkers are known in the art. These include: succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl(4-iodoacetyl) aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC);4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-tolune (SMPT),N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), succinimidyl6-[3-(2-pyridyldithio) propionate] hexanoate (LC-SPDP). Thosecross-linking agents having N-hydroxysuccinimide moieties can beobtained as the N-hydroxysulfosuccinimide analogs, which generally havegreater water solubility. In addition, those cross-linking agents havingdisulfide bridges within the linking chain can be synthesized instead asthe alkyl derivatives so as to reduce the amount of linker cleavage invivo.

In addition to the heterobifunctional cross-linkers, there exists anumber of other cross-linking agents including homobifunctional andphotoreactive cross-linkers. Disuccinimidyl suberate (DSS),bismaleimidohexane (BMH) and dimethylpimelimidate-2 HCl (DMP) areexamples of useful homobifunctional cross-linking agents, andbis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED) andN-succinimidyl-6(4′-azido-2′-nitrophenyl-amino)hexanoate (SANPAH) areexamples of useful photoreactive cross-linkers for use in thisinvention. For a recent review of protein coupling techniques, see Meanset al. (1990) Bioconjugate Chemistry 1:2-12, incorporated by referenceherein.

One particularly useful class of heterobifunctional cross-linkers,included above, contain the primary amine reactive group,N-hydroxysuccinimide (NHS), or its water soluble analogN-hydroxysulfosuccinimide (sulfo-NHS). Primary amines (lysine epsilongroups) at alkaline pH's are unprotonated and react by nucleophilicattack on NHS or sulfo-NHS esters. This reaction results in theformation of an amide bond, and release of NHS or sulfo-NHS as aby-product.

Another reactive group useful as part of a heterobifunctionalcross-linker is a thiol reactive group. Common thiol reactive groupsinclude maleimides, halogens, and pyridyl disulfides. Maleimides reactspecifically with free sulfhydryls (cysteine residues) in minutes, underslightly acidic to neutral (pH 6.5-7.5) conditions. Halogens (iodoacetylfunctions) react with —SH groups at physiological pH's. Both of thesereactive groups result in the formation of stable thioether bonds.

The third component of the heterobifunctional cross-linker is the spacerarm or bridge. The bridge is the structure that connects the tworeactive ends. The most apparent attribute of the bridge is its effecton steric hindrance. In some instances, a longer bridge can more easilyspan the distance necessary to link two complex biomolecules. Forinstance, SMPB has a span of 14.5 angstroms.

Preparing protein—protein conjugates using heterobifunctional reagentsis a two-step process involving the amine reaction and the sulfhydrylreaction. For the first step, the amine reaction, the protein chosenshould contain a primary amine. This can be lysine epsilon amines or aprimary alpha amine found at the N-terminus of most proteins. Theprotein should not contain free sulfhydryl groups. In cases where bothproteins to be conjugated contain free sulfhydryl groups, one proteincan be modified so that all sulfhydryls are blocked using for instance,N-ethylmaleimide (see Partis et al. (1983) J. Pro. Chem. 2:263,incorporated by reference herein). Ellman's Reagent can be used tocalculate the quantity of sulfhydryls in a particular protein (see forexample Ellman et al. (1958) Arch. Biochem. Biophys. 74:443 and Riddleset al. (1979) Anal. Biochem. 94:75, incorporated by reference herein).

The reaction buffer should be free of extraneous amines and sulfhydryls.The pH of the reaction buffer should be 7.0-7.5. This pH range preventsmaleimide groups from reacting with amines, preserving the maleimidegroup for the second reaction with sulfhydryls.

The NHS-ester containing cross-linkers have limited water solubility.They should be dissolved in a minimal amount of organic solvent (DMF orDMSO) before introducing the cross-linker into the reaction mixture. Thecross-linker/solvent forms an emulsion which will allow the reaction tooccur.

The sulfo-NHS ester analogs are more water soluble, and can be addeddirectly to the reaction buffer. Buffers of high ionic strength shouldbe avoided, as they have a tendency to “salt out” the sulfo-NHS esters.To avoid loss of reactivity due to hydrolysis, the cross-linker is addedto the reaction mixture immediately after dissolving the proteinsolution.

The reactions can be more efficient in concentrated protein solutions.The more alkaline the pH of the reaction mixture, the faster the rate ofreaction. The rate of hydrolysis of the NHS and sulfo-NHS esters willalso increase with increasing pH. Higher temperatures will increase thereaction rates for both hydrolysis and acylation.

Once the reaction is completed, the first protein is now activated, witha sulfhydryl reactive moiety. The activated protein may be isolated fromthe reaction mixture by simple gel filtration or dialysis. To carry outthe second step of the cross-linking, the sulfhydryl reaction, thelipophilic group chosen for reaction with maleimides, activatedhalogens, or pyridyl disulfides must contain a free sulfhydryl.Alternatively, a primary amine may be modified with to add a sulfhydryl

In all cases, the buffer should be degassed to prevent oxidation ofsulfhydryl groups. EDTA may be added to chelate any oxidizing metalsthat may be present in the buffer. Buffers should be free of anysulfhydryl containing compounds.

Maleimides react specifically with —SH groups at slightly acidic toneutral pH ranges (6.5-7.5). A neutral pH is sufficient for reactionsinvolving halogens and pyridyl disulfides. Under these conditions,maleimides generally react with —SH groups within a matter of minutes.Longer reaction times are required for halogens and pyridyl disulfides.

The first sulfhydryl reactive-protein prepared in the amine reactionstep is mixed with the sulfhydryl-containing lipophilic group under theappropriate buffer conditions. The conjugates can be isolated from thereaction mixture by methods such as gel filtration or by dialysis.

Exemplary activated lipophilic moieties for conjugation include:N-(1-pyrene)maleimide; 2,5-dimethoxystilbene-4′-maleimide,eosin-5-maleimide; fluorescein-5-maleimide;N-(4-(6-dimethylamino-2-benzofuranyl)phenyl)maleimide;benzophenone-4-maleimide; 4-dimethylaminophenylazophenyl-4′-maleimide(DABMI), tetramethylrhodamine-5-maleimide,tetramethylrhodamine-6-maleimide, Rhodamine RedTM C2 maleimide.N-(5-aminopentyl)maleimide, trifluoroacetic acid salt,N-(2-aminoethyl)maleimide, trifluoroacetic acid salt, Oregon GreenTM 488maleimide,N-(2-((2-(((4-azido-2,3,5,6-tetrafluoro)benzoyl)amino)ethyl)dithio)ethyl)maleimide(TFPAM-SS1), 2-(1-(3-dimethylaminopropyl)-indol-3-yl)-3-(indol-3-yl)maleimide (bisindolylmaleimide; GF 109203X), BODIPY® FLN-(2-aminoethyl)maleimide,N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM), AlexaTM 488C5 maleimide, AlexaTM 594 C5 maleimide, sodiumsaltN-(1-pyrene)maleimide, 2,5-dimethoxystilbene-4′-maleimide,eosin-5-maleimide, fluorescein-5-maleimide,N-(4-(6-dimethylamino-2-benzofuranyl)phenyl)maleimide,benzophenone-4-maleimide, 4-dimethylaminophenylazophenyl-4-maleimide,1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridiniummethanesulfonate, tetramethylrhodamine-5-maleimide,tetramethylrhodamine-6-maleimide, Rhodamine RedTM C2 maleimide,N-(5-aminopentyl)maleimide, N-(2-aminoethyl)maleimide,N-(2-((2-(((4-azido-2,3,5,6-tetrafluoro)benzoyl)amino)ethyl)dithio)ethyl)maleimide,2-(1-(3-dimethylaminopropyl)-indol-3-yl)-3-(indol-3-yl)maleimide,N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM),11H-Benzo[a]fluorene, Benzo[a]pyrene.

In one embodiment, the hedgehog polypeptide can be derivatived usingpyrene maleimide, which can be purchased from Molecular Probes (Eugene,Oreg.), e.g., N-(1-pyrene)maleimide or 1-pyrenemethyl iodoacetate (PMIAester).

For those embodiments wherein the hydophobic moiety is a polypeptide,the modified hedgehog polypeptide of this invention can be constructedas a fusion protein, containing the hedgehog polypeptide and thehydrophobic moiety as one contiguous polypeptide chain.

In certain embodiments, the lipophilic moiety is an amphipathicpolypeptide, such as magainin, cecropin, attacin, melittin, gramicidinS, alpha-toxin of Staph. aureus, alamethicin or a synthetic amphipathicpolypeptide. Fusogenic coat proteins from viral particles can also be aconvenient source of amphipathic sequences for the subject hedgehogproteins

Moreover, mutagenesis can be used to create modified hh polypeptides,e.g., for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo). Such modified peptides can beproduced, for instance, by amino acid substitution, deletion, oraddition. Modified hedgehog polypeptides can also include those withaltered post-translational processing relative to a naturally occurringhedgehog protein, e.g., altered glycosylation, cholesterolization,prenylation and the like.

In one embodiment, the hedgehog therapeutic is a polypeptide encodableby a nucleotide sequence that hybridizes under stringent conditions to ahedgehog coding sequence represented in one or more of SEQ ID Nos: 1-7.Appropriate stringency conditions which promote DNA hybridization, forexample. 6.0×sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C.

As described in the literature, genes for other hedgehog proteins, e.g.,from other animals, can be obtained from mRNA or genomic DNA samplesusing techniques well known in the art. For example, a eDNA encoding ahedgehog protein can be obtained by isolating total mRNA from a cell,e.g. a mammalian cell, e.g. a human cell, including embryonic cells.Double stranded cDNAs can then be prepared from the total mRNA, andsubsequently inserted into a suitable plasmid or bacteriophage vectorusing any one of a number of known techniques. The gene encoding ahedgehog protein can also be cloned using established polymerase chainreaction techniques.

Preferred nucleic acids encode a hedgehog polypeptide comprising anamino acid sequence at least 60% homologous or identical, morepreferably 70% homologous or identical, and most preferably 80%homologous or identical with an amino acid sequence selected from thegroup consisting of SEQ ID Nos:8-14. Nucleic acids which encodepolypeptides at least about 90%, more preferably at least about 95%, andmost preferably at least about 98-99% homology or identity with an aminoacid sequence represented in one of SEQ ID Nos:8-14 are also within thescope of the invention.

In addition to native hedgehog proteins, hedgehog polypeptides preferredby the present invention are at least 60% homologous or identical, morepreferably 70% homologous or identical and most preferably 80%homologous or identical with an amino acid sequence represented by anyof SEQ ID Nos:8-14. Polypeptides which are at least 90%, more preferablyat least 95%, and most preferably at least about 98-99% homologous oridentical with a sequence selected from the group consisting of SEQ IDNos:8-14 are also within the scope of the invention. The onlyprerequisite is that the hedgehog polypeptide is capable of modulatingthe growth of epithelial cells.

The term “recombinant protein” refers to a polypeptide of the presentinvention which is produced by recombinant DNA techniques, whereingenerally, DNA encoding a hedgehog polypeptide is inserted into asuitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein. Moreover, the phrase “derivedfrom”, with respect to a recombinant hedgehog gene, is meant to includewithin the meaning of “recombinant protein” those proteins having anamino acid sequence of a native hedgehog protein, or an amino acidsequence similar thereto which is generated by mutations includingsubstitutions and deletions (including truncation) of a naturallyoccurring form of the protein.

The method of the present invention can also be carried out usingvariant forms of the naturally occurring hedgehog polypeptides, e.g.,mutational variants.

As is known in the art, hedgehog polypeptides can be produced bystandard biological techniques or by chemical synthesis. For example, ahost cell transfected with a nucleic acid vector directing expression ofa nucleotide sequence encoding the subject polypeptides can be culturedunder appropriate conditions to allow expression of the peptide tooccur. The polypeptide hedgehog may be secreted and isolated from amixture of cells and medium containing the recombinant hedgehogpolypeptide. Alternatively, the peptide may be retained cytoplasmicallyby removing the signal peptide sequence from the recombinant hedgehoggene and the cells harvested, Iysed and the protein isolated. A cellculture includes host cells, media and other byproducts. Suitable mediafor cell culture are well known in the art. The recombinant hedgehogpolypeptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for such peptide. In a preferred embodiment, therecombinant hedgehog polypeptide is a fusion protein containing a domainwhich facilitates its purification, such as an hedgehog/GST fusionprotein. The host cell may be any prokaryotic or eukaryotic cell.

Recombinant hedgehog genes can be produced by ligating nucleic acidencoding an hedgehog protein, or a portion thereof, into a vectorsuitable for expression in either prokaryotic cells, eukaryotic cells,or both. Expression vectors for production of recombinant forms of thesubject hedgehog polypeptides include plasmids and other vectors. Forinstance, suitable vectors for the expression of a hedgehog polypeptideinclude plasmids of the types: pBR322-derived plasmids, pEMBL-derivedplasmids., pEX-derived plasmids, pBTac-derived plasmids and pUC-derivedplasmids for expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance. YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due to the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, an hedgehog polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of one of the hedgehog genes represented in SEQ IDNos: 1-7.

The preferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989) Chapters 16 and 17.

In some instances, it may be desirable to express the recombinanthedgehog polypeptide by the use of a baculovirus expression system.Examples of such baculovirus expression systems include pVL-derivedvectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors(such as pAcUW1), and pBlueBac-derived vectors (such as the β-galcontaining pBlueBac III).

When it is desirable to express only a portion of an hedgehog protein,such as a form lacking a portion of the N-terminus, i.e. a truncationmutant which lacks the signal peptide, it may be necessary to add astart codon (ATG) to the oligonucleotide fragment containing the desiredsequence to be expressed. It is well known in the art that a methionineat the N-terminal position can be enzymatically cleaved by the use ofthe enzyme methionine aminopeptidase (MAP). MAP has been cloned from E.coli (Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing hedgehog-derived polypeptides in ahost which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or invitro by use of purified MAP (e.g., procedure of Miller et al., supra).

Alternatively, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different polypeptide. It is widely appreciated that fusionproteins can also facilitate the expression of proteins, andaccordingly, can be used in the expression of the hedgehog polypeptidesof the present invention. For example, hedgehog polypeptides can begenerated as glutathione-S-transferase (GST-fusion) proteins. SuchGST-fusion proteins can enable easy purification of the hedgehogpolypeptide, as for example by the use of glutathione-derivatizedmatrices (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. (N.Y.: John Wiley & Sons, 1991)). In another embodiment,a fusion gene coding for a purification leader sequence, such as apoly-(His)/enterokinase cleavage site sequence, can be used to replacethe signal sequence which naturally occurs at the N-terminus of thehedgehog protein (e.g. of the pro-form, in order to permit purificationof the poly(His)-hedgehog protein by affinity chromatography using aNi²⁺ metal resin. The purification leader sequence can then besubsequently removed by treatment with enterokinase (e.g., see Hochuliet al. (1987) J. Chromatography 411:177; and Janknecht et al. PNAS88:8972).

Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

Hedgehog polypeptides may also be chemically modified to create hedgehogderivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, cholesterol, isoprenoids,lipids, phosphate, acetyl groups and the like. Covalent derivatives ofhedgehog proteins can be prepared by linking the chemical moieties tofunctional groups on amino acid sidechains of the protein or at theN-terminus or at the C-terminus of the polypeptide.

For instance, hedgehog proteins can be generated to include a moiety,other than sequence naturally associated with the protein, that binds acomponent of the extracellular matrix and enhances localization of theanalog to cell surfaces. For example, sequences derived from thefibronectin “type-III repeat”, such as a tetrapeptide sequence R-G-D-S(Pierschbacher et al. (1984) Nature 309:30-3; and Kornblihtt et al.(1985) EMBO 4:1755-9) can be added to the hedgehog polypeptide tosupport attachment of the chimeric molecule to a cell through bindingECM components (Ruoslahti et al. (1987) Science 238:491-497;Pierschbacheret al. (1987) J. Biol. Chem. 262:17294-8.; Hynes (1987)Cell 48:549-54; and Hynes (1992) Cell 69:11-25).

In a preferred embodiment, the hedgehog polypeptide is isolated from, oris otherwise substantially free of, other cellular proteins, especiallyother extracellular or cell surface associated proteins which maynormally be associated with the hedgehog polypeptide, unless provided inthe form of fusion protein with the hedgehog polypeptide. The term“substantially free of other cellular or extracellular proteins” (alsoreferred to herein as “contaminating proteins”) or “substantially purepreparations” or “purified preparations” are defined as encompassingpreparations of hedgehog polypeptides having less than 20% (by dryweight) contaminating protein, and preferably having less than 5%contaminating protein. By “purified”, it is meant that the indicatedmolecule is present in the substantial absence of other biologicalmacromolecules, such as other proteins. The term “purified” as usedherein preferably means at least 80% by dry weight, more preferably inthe range of 95-99% by weight, and most preferably at least 99.8% byweight, of biological macromolecules of the same type present (butwater, buffers, and other small molecules, especially molecules having amolecular weight of less than 5000, can be present). The term “pure” asused herein preferably has the same numerical limits as “purified”immediately above.

As described above for recombinant polypeptides, isolated hedgehogpolypeptides can include all or a portion of the amino acid sequencesrepresented in any of SEQ ID Nos:10-18 or 20, or a homologous sequencethereto. Preferred fragments of the subject hedgehog proteins correspondto the N-terminal and C-terminal proteolytic fragments of the matureprotein. Bioactive fragments of hedgehog polypeptides are described ingreat detail in PCT publications WO 95/18856 and WO 96/17924.

With respect to bioctive fragments of hedgehog polypeptide, preferredhedgehog therapeutics include at least 50 (contiguous) amino acidresidues of a hedgehog polypeptide, more preferably at least 100(contiguous), and even more preferably at least 150 (contiguous)residues.

Another preferred hedgehog polypeptide which can be included in thehedgehog therapeutic is an N-terminal fragment of the mature proteinhaving a molecular weight of approximately 19 kDa.

Preferred human hedgehog proteins include N-terminal fragmentscorresponding approximately to residues 24-197 of SEQ ID No. 15, 28-202of SEQ ID No. 16, and 23-198 of SEQ ID No. 17. By “correspondingapproximately” it is meant that the sequence of interest is at most 20amino acid residues different in length to the reference sequence,though more preferably at most 5, 10 or 15 amino acid different inlength.

As described above for recombinant polypeptides, isolated hedgehogpolypeptides can include all or a portion of the amino acid sequencesrepresented in SEQ ID No:8, SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQID No:12, SEQ ID No:13 or SEQ ID No:14, or a homologous sequencethereto. Preferred fragments of the subject hedgehog proteins correspondto the N-terminal and C-terminal proteolytic fragments of the matureprotein. Bioactive fragments of hedgehog polypeptides are described ingreat detail in PCT publications WO 95/18856 and WO 96/17924.

Still other preferred hedgehog polypeptides includes an amino acidsequence represented by the formula A-B wherein: (i) A represents all orthe portion of the amino acid sequence designated by residues 1-168 ofSEQ ID No:21; and B represents at least one amino acid residue of theamino acid sequence designated by residues 169-221 of SEQ ID No:21; (ii)A represents all or the portion of the amino acid sequence designated byresidues 24-193 of SEQ ID No:15; and B represents at least one aminoacid residue of the amino acid sequence designated by residues 194-250of SEQ ID No:15; (iii) A represents all or the portion of the amino acidsequence designated by residues 25-193 of SEQ ID No:13; and B representsat least one amino acid residue of the amino acid sequence designated byresidues 194-250 of SEQ ID No:13; (iv) A represents all or the portionof the amino acid sequence designated by residues 23-193 of SEQ IDNo:11; and B represents at least one amino acid residue of the aminoacid sequence designated by residues 194-250 of SEQ ID No:11; (v) Arepresents all or the portion of the amino acid sequence designated byresidues 28-197 of SEQ ID No:12; and B represents at least one aminoacid residue of the amino acid sequence designated by residues 198-250of SEQ ID No:12; (vi) A represents all or the portion of the amino acidsequence designated by residues 29-197 of SEQ ID No:16; and B representsat least one amino acid residue of the amino acid sequence designated byresidues 198-250 of SEQ ID No:16; or (vii) A represents all or theportion of the amino acid sequence designated by residues 23-193 of SEQID No. 17, and B represents at least one amino acid residue of the aminoacid sequence designated by residues 194-250 of SEQ ID No. 17. Incertain preferred embodiments, A and B together represent a contiguouspolypeptide sequence designated sequence, A represents at least 25, 50,75, 100, 125 or 150 (contiguous) amino acids of the designated sequence,and B represents at least 5, 10, or 20 (contiguous) amino acid residuesof the amino acid sequence designated by corresponding entry in thesequence listing, and A and B together preferably represent a contiguoussequence corresponding to the sequence listing entry. Similar fragmentsfrom other hedgehog also contemplated, e.g., fragments which correspondto the preferred fragments from the sequence listing entries which areenumerated above. In preferred embodiments, the hedgehog polypeptideincludes a C-terminal glycine (or other appropriate residue) which isderivatized with a cholesterol.

Isolated peptidyl portions of hedgehog proteins can be obtained byscreening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, a hedgehog polypeptide of the present inventionmay be arbitrarily divided into fragments of desired length with nooverlap of the fragments, or preferably divided into overlappingfragments of a desired length. The fragments can be produced(recombinantly or by chemical synthesis) and tested to identify thosepeptidyl fragments which can function as either agonists or antagonistsof a wild-type (e.g., “authentic”) hedgehog protein. For example, Románet al. (1994) Eur J Biochem 222:65-73 describe the use ofcompetitive-binding assays using short, overlapping synthetic peptidesfrom larger proteins to identify binding domains.

The recombinant hedgehog polypeptides of the present invention alsoinclude homologs of the authentic hedgehog proteins, such as versions ofthose protein which are resistant to proteolytic cleavage, as forexample, due to mutations which alter potential cleavage sequences orwhich inactivate an enzymatic activity associated with the protein.Hedgehog homologs of the present invention also include proteins whichhave been post-translationally modified in a manner different than theauthentic protein. Exemplary derivatives of hedgehog proteins includepolypeptides which lack N-glycosylation sites (e.g. to produce anunglycosylated protein), which lack sites for cholesterolization, and/orwhich lack N-terminal and/or C-terminal sequences.

Modification of the structure of the subject hedgehog polypeptides canalso be for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo). Such modified peptides, when designedto retain at least one activity of the naturally-occurring form of theprotein, are considered functional equivalents of the hedgehogpolypeptides described in more detail herein. Such modified peptides canbe produced, for instance, by amino acid substitution, deletion, oraddition.

It is well known in the art that one could reasonably expect thatcertain isolated replacements of amino acids, e.g., replacement of anamino acid residue with another related amino acid (i.e. isostericand/or isoelectric mutations), can be carried out without major effecton the biological activity of the resulting molecule. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidsare can be divided into four families: (1) acidic=aspartate, glutamate;(2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (see, for example, Biochemistry, 2nd ed., Ed. by L. Stryer,W H Freeman and Co.: 1981). Whether a change in the amino acid sequenceof a peptide results in a functional hedgehog homolog (e.g. functionalin the sense that it acts to mimic or antagonize the wild-type form) canbe readily determined by assessing the ability of the variant peptide toproduce a response in cells in a fashion similar to the wild-typeprotein, or competitively inhibit such a response. Polypeptides in whichmore than one replacement has taken place can readily be tested in thesame manner.

It is specifically contemplated that the methods of the presentinvention can be carried using homologs of naturally occurring hedgehogproteins. In one embodiment, the invention contemplates using hedgehogpolypeptides generated by combinatorial mutagenesis. Such methods, asare known in the art, are convenient for generating both point andtruncation mutants, and can be especially useful for identifyingpotential variant sequences (e.g. homologs) that are functional inbinding to a receptor for hedgehog proteins. The purpose of screeningsuch combinatorial libraries is to generate, for example, novel hedgehoghomologs which can act as either agonists or antagonist. To illustrate,hedgehog homologs can be engineered by the present method to providemore efficient binding to a cognate receptor, such as patched, yet stillretain at least a portion of an activity associated with hedgehog. Thus,combinatorially-derived homologs can be generated to have an increasedpotency relative to a naturally occurring form of the protein. Likewise,hedgehog homologs can be generated by the present combinatorial approachto act as antagonists, in that they are able to mimic, for example,binding to other extracellular matrix components (such as receptors),yet not induce any biological response, thereby inhibiting the action ofauthentic hedgehog or hedgehog agonists. Moreover, manipulation ofcertain domains of hedgehog by the present method can provide domainsmore suitable for use in fusion proteins, such as one that incorporatesportions of other proteins which are derived from the extracellularmatrix and/or which bind extracellular matrix components.

To further illustrate the state of the art of combinatorial mutagenesis,it is noted that the review article of Gallop et al. (1994) J Med Chem37:1233 describes the general state of the art of combinatoriallibraries as of the earlier 1990's. In particular, Gallop et al state atpage 1239 “[s]creening the analog libraries aids in determining theminimum size of the active sequence and in identifying those residuescritical for binding and intolerant of substitution”. In addition, theLadner et al. PCT publication WO90/02809, the Goeddel et al. U.S. Pat.No. 5,223,408, and the Markland et al. PCT publication WO92/15679illustrate specific techniques which one skilled in the art couldutilize to generate libraries of hedgehog variants which can be rapidlyscreened to identify variants/fragments which retained a particularactivity of the hedgehog polypeptides. These techniques are exemplary ofthe art and demonstrate that large libraries of relatedvariants/truncants can be generated and assayed to isolate particularvariants without undue experimentation. Gustin et al. (1993) Virology193:653, and Bass et al. (1990) Proteins: Structure, Function andGenetics 8:309-314 also describe other exemplary techniques from the artwhich can be adapted as means for generating mutagenic variants ofhedgehog polypeptides.

Indeed, it is plain from the combinatorial mutagenesis art that largescale mutagenesis of hedgehog proteins, without any preconceived ideasof which residues were critical to the biological function, and generatewide arrays of variants having equivalent biological activity. Indeed,it is the ability of combinatorial techniques to screen billions ofdifferent variants by high throughout analysis that removes anyrequirement of a priori understanding or knowledge of critical residues.

To illustrate, the amino acid sequences for a population of hedgehoghomologs or other related proteins are aligned, preferably to promotethe highest homology possible. Such a population of variants caninclude, for example, hedgehog homologs from one or more species. Aminoacids which appear at each position of the aligned sequences areselected to create a degenerate set of combinatorial sequences. In apreferred embodiment, the variegated library of hedgehog variants isgenerated by combinatorial mutagenesis at the nucleic acid level, and isencoded by a variegated gene library. For instance, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential hedgehog sequencesare expressible as individual polypeptides, or alternatively, as a setof larger fusion proteins (e.g. for phage display) containing the set ofhedgehog sequences therein.

As illustrated in PCT publication WO 95/18856, to analyze the sequencesof a population of variants, the amino acid sequences of interest can bealigned relative to sequence homology. The presence or absence of aminoacids from an aligned sequence of a particular variant is relative to achosen consensus length of a reference sequence, which can be real orartificial.

In an illustrative embodiment, alignment of exons 1, 2 and a portion ofexon 3 encoded sequences (e.g. the N-terminal approximately 221 residuesof the mature protein) of each of the Shh clones produces a degenerateset of Shh polypeptides represented by the general formula:

C-G-P-G-R-G-X(1)-G-X(2)-R-R-H-P-K-K-L-T-P-L-A-Y-K-Q-F-I-P-N-V-A-E- (SEQID No:21K-T-L-G-A-S-G-R-Y-E-G-K-I-X(3)-R-N-S-E-R-F-K-E-L-T-P-N-Y-N-P-D-I-I-F-K-D-E-E-N-T-G-A-D-R-L-M-T-Q-R-C-K-D-K-L-N-X(4)-L-A-I-S-V-M-N-X(5)-W-P-G-V-X(6)-L-R-V-T-E-G-W-D-E-D-G-H-H-X(7)-E-E-S-L-H-Y-E-G-R-A-V-D-I-T-T-S-D-R-D-X(8)-S-K-Y-G-X(9)-L-X(10)-R-L-A-V-E-A-G-F-D-W-V-Y-Y-E-S-K-A-H-I-H-C-S-V-K-A-E-N-S-V-A-A-K-S-G-G-C-F-P-G-S-A-X(11)-V-X(12)-L-X(13)-X(14)-G-G-X(15)-K-X-(16)-V-K-D-L-X(17)-P-G-D-X(18)-V-L-A-A-D-X(19)-X(20)-G-X(21)-L-X(22)-X(23)-S-D-F-X(24)-X(25)-F-X(26)-D- R

wherein each of the degenerate positions “X” can be an amino acid whichoccurs in that position in one of the human, mouse, chicken or zebrafishShh clones, or, to expand the library, each X can also be selected fromamongst amino acid residue which would be conservative substitutions forthe amino acids which appear naturally in each of those positions. Forinstance, Xaa(1) represents Gly, Ala, Val, Leu, Ile, Phe, Tyr or Trp;Xaa(2) represents Arg, His or Lys; Xaa(3) represents Gly, Ala, Val, Leu,Ile, Ser or Thr; Xaa(4) represents Gly, Ala, Val, Leu, Ile, Ser or Thr;Xaa(5) represents Lys, Arg, His, Asn or Gln; Xaa(6) represents Lys, Argor His; Xaa(7) represents Ser, Thr, Tyr, Trp or Phe; Xaa(8) representsLys, Arg or His; Xaa(9) represents Met, Cys, Ser or Thr; Xaa(10)represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(11) represents Leu,Val, Met, Thr or Ser; Xaa(12) represents His, Phe, Tyr, Ser, Thr, Met orCys; Xaa(13) represents Gln, Asn, Glu, or Asp; Xaa(14) represents His,Phe, Tyr, Thr, Gln, Asn, Glu or Asp; Xaa(15) represents Gln, Asn, Glu,Asp, Thr, Ser, Met or Cys; Xaa(16) represents Ala, Gly, Cys, Leu, Val orMet; Xaa(17) represents Arg, Lys, Met, Ile, Asn, Asp, Glu, Gln, Ser, Thror Cys; Xaa(18) represents Arg, Lys, Met or Ile; Xaa(19) represents Ala,Gly, Cys, Asp, Glu, Gln, Asn, Ser, Thr or Met; Xaa(20) represents Ala,Gly, Cys, Asp, Asn, Glu or Gln; Xaa(21) represents Arg, Lys, Met, Ile,Asn, Asp, Glu or Gln; Xaa(22) represent Leu, Val, Met or Ile; Xaa(23)represents Phe, Tyr, Thr, His or Trp; Xaa(24) represents Ile, Val, Leuor Met; .Xaa(25) represents Met, Cys. Ile, Leu, Val, Thr or Ser; Xaa(26)represents Leu, Val, Met, Thr or Ser. In an even more expansive library,each X can be selected from any amino acid.

In similar fashion, alignment of each of the human, mouse, chicken andzebrafish hedgehog clones, can provide a degenerate polypeptide sequencerepresented by the general formula:

C-G-P-G-R-G-X(1)-X(2)-X(3)-R-R-X(4)-X(5)-X(6)-P-K-X(7)-L-X(8)-P-L-X(9)-(SEQ ID No:22Y-K-Q-F-X(10)-P-X(11)-X(12)-X(13)-E-X(14)-T-L-G-A-S-G-X(15)-X(16)-E-G-X(17)-X(18)-X(19)-R-X(20)-S-E-R-F-X(21)-X(22)-L-T-P-N-Y-N-P-D-I-I-F-K-D-E-E-N-X(23)-G-A-D-R-L-M-T-X(24)-R-C-K-X(25)-X(26)-X(27)-N-X(28)-L-A-I-S-V-M-N-X(29)-W-P-G-V-X(30)-L-R-V-T-E-G-X(31)-D-E-D-G-H-H-X(32)-X(33)-X(34)-S-L-H-Y-E-G-R-A-X(35)-D-I-T-T-S-D-R-D-X(36)-X(37)-K-Y-G-X(38)-L-X(39)-R-L-A-V-E-A-G-F-D-W-V-Y-Y-E-S-X(40)-X(41)-H-X(42)-H-X(43)-S-V-K-X(44)-X(45)

wherein, as above, each of the degenerate positions “X” can be an aminoacid which occurs in a corresponding position in one of the wild-typeclones, and may also include amino acid residue which would beconservative substitutions, or each X can be any amino acid residue. Inan exemplary embodiment, Xaa(1) represents Gly, Ala, Val, Leu, Ile, Pro,Phe or Tyr; Xaa(2) represents Gly, Ala, Val, Leu or Ile; Xaa(3)represents Gly, Ala, Val, Leu, Ile, Lys, His or Arg; Xaa(4) representsLys, Arg or His; Xaa(5) represents Phe, Trp, Tyr or an amino acid gap;Xaa(6) represents Gly, Ala, Val, Leu, Ile or an amino acid gap; Xaa(7)represents Asn, Gln, His, Arg or Lys; Xaa(8) represents Gly, Ala, Val,Leu, Ile, Ser or Thr; Xaa(9) represents Gly, Ala, Val, Leu, Ile, Ser orThr; Xaa(10) represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(11)represents Ser, Thr, Gln or Asn; Xaa(12) represents Met, Cys, Gly, Ala,Val, Leu, Ile, Ser or Thr; Xaa(13) represents Gly, Ala, Val, Leu, Ile orPro; Xaa(14) represents Arg, His or Lys; Xaa(15) represents Gly, Ala,Val, Leu, Ile, Pro, Arg, His or Lys; Xaa(16) represents Gly, Ala, Val,Leu, Ile, Phe or Tyr; Xaa(17) represents Arg, His or Lys; Xaa(18)represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(19) represents Thror Ser; Xaa(20) represents Gly, Ala, Val, Leu, Ile, Asn or Gln; Xaa(21)represents Arg, His or Lys; Xaa(22) represents Asp or Glu; Xaa(23)represents Ser or Thr; Xaa(24) represents Glu, Asp, Gln or Asn; Xaa(25)represents Glu or Asp; Xaa(26) represents Arg, His or Lys; Xaa(27)represents Gly, Ala, Val, Leu or Ile; Xaa(28) represents Gly, Ala, Val,Leu, Ile, Thr or Ser; Xaa(29) represents Met, Cys, Gln, Asn, Arg, Lys orHis; Xaa(30) represents Arg, His or Lys; Xaa(31) represents Trp, Phe,Tyr, Arg, His or Lys; Xaa(32) represents Gly, Ala, Val, Leu, Ile Ser,Thr, Tyr or Phe; Xaa(33) represents Gln, Asn, Asp or Glu; Xaa(34)represents Asp or Glu: Xaa(35) represents Gly, Ala, Val, Leu, or Ile;Xaa(36) represents Arg, His or Lys; Xaa(37) represents Asn, Gln, Thr orSer; Xaa(38) represents Gly, Ala, Val, Leu, Ile, Ser, Thr, Met or Cys;Xaa(39) represents Gly, Ala, Val, Leu, Ile, Thr or Ser; Xaa(40)represents Arg, His or Lys; Xaa(41) represents Asn, Gln, Gly, Ala, Val,Leu or Ile; Xaa(42) represents Gly, Ala, Val, Leu or Ile; Xaa(43)represents Gly, Ala, Val, Leu, Ile, Ser, Thr or Cys; Xaa(44) representsGly, Ala, Val, Leu, Ile, Thr or Ser; and Xaa(45) represents Asp or Glu.

There are many ways by which the library of potential hedgehog homologscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into anappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential hedgehog sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, S A(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevierpp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al. (1990) Science 249:386-390;Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S.Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations, and forscreening cDNA libraries for gene products having a certain property.Such techniques will be generally adaptable for rapid screening of thegene libraries generated by the combinatorial mutagenesis of hedgehoghomologs. The most widely used techniques for screening large genelibraries typically comprises cloning the gene library into replicableexpression vectors, transforming appropriate cells with the resultinglibrary of vectors, and expressing the combinatorial genes underconditions in which detection of a desired activity facilitatesrelatively easy isolation of the vector encoding the gene whose productwas detected. Each of the illustrative assays described below areamenable to high through-put analysis as necessary to screen largenumbers of degenerate hedgehog sequences created by combinatorialmutagenesis techniques.

In one embodiment, the combinatorial library is designed to be secreted(e.g. the polypeptides of the library all include a signal sequence butno transmembrane or cytoplasmic domains), and is used to transfect aeukaryotic cell that can be co-cultured with epithelial stem cells. Afunctional hedgehog protein secreted by the cells expressing thecombinatorial library will diffuse to neighboring epithelial cells andinduce a particular biological response, such as proliferation. Thepattern of detection of proliferation will resemble a gradient function,and will allow the isolation (generally after several repetitive roundsof selection) of cells producing hedgehog homologs active asproliferative agents with respect to epithelial cells. Likewise,hedgehog antagonists can be selected in similar fashion by the abilityof the cell producing a functional antagonist to protect neighboringcells (e.g., to inhibit proliferation) from the effect of wild-typehedgehog added to the culture media.

To illustrate, target epithelial cells are cultured in 24-wellmicrotitre plates. Other eukaryotic cells are transfected with thecombinatorial hedgehog gene library and cultured in cell culture inserts(e.g. Collaborative Biomedical Products, Catalog #40446) that are ableto fit into the wells of the microtitre plate. The cell culture insertsare placed in the wells such that recombinant hedgehog homologs secretedby the cells in the insert can diffuse through the porous bottom of theinsert and contact the target cells in the microtitre plate wells. Aftera period of time sufficient for functional forms of a hedgehog proteinto produce a measurable response in the target cells, such asproliferation, the inserts are removed and the effect of the varianthedgehog proteins on the target cells determined. Cells from the insertscorresponding to wells which score positive for activity can be splitand re-cultured on several inserts, the process being repeated until theactive clones are identified.

In yet another screening assay, the candidate hedgehog gene products aredisplayed on the surface of a cell or viral particle, and the ability ofparticular cells or viral particles to associate with a hedgehog-bindingmoiety (such as the patched protein or other hedgehog receptor) via thisgene product is detected in a “panning assay”. Such panning steps can becarried out on cells cultured from embryos. For instance, the genelibrary can be cloned into the gene for a surface membrane protein of abacterial cell, and the resulting fusion protein detected by panning(Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology9:1370-1371; and Goward et al. (1992) TIBS 18:136-140). In a similarfashion, fluorescently labeled molecules which bind hedgehog can be usedto score for potentially functional hedgehog homologs. Cells can bevisually inspected and separated under a fluorescence microscope, or,where the morphology of the cell permits, separated by afluorescence-activated cell sorter.

In an alternate embodiment, the gene library is expressed as a fusionprotein on the surface of a viral particle. For instance, in thefilamentous phage system, foreign peptide sequences can be expressed onthe surface of infectious phage, thereby conferring two significantbenefits. First, since these phage can be applied to affinity matricesat very high concentrations, large number of phage can be screened atone time. Second, since each infectious phage displays the combinatorialgene product on its surface, if a particular phage is recovered from anaffinity matrix in low yield, the phage can be amplified by anotherround of infection. The group of almost identical E. coli filamentousphages M13, fd, and f1 are most often used in phage display libraries,as either of the phage gIII or gVIII coat proteins can be used togenerate fusion proteins without disrupting the ultimate packaging ofthe viral particle (Ladner et al. PCT publication WO 90/02909; Garrardet al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007-16010; Griffths et al. (1993) EMBO J 12:725-734; Clackson etal. (1991) Nature 352:624-628: and Barbas et al. (1992) PNAS89:4457-4461).

In an illustrative embodiment, the recombinant phage antibody system(RPAS, Pharamacia Catalog number 27-9400-01) can be easily modified foruse in expressing and screening hedgehog combinatorial libraries. Forinstance, the pCANTAB 5 phagemid of the RPAS kit contains the gene whichencodes the phage gIII coat protein. The hedgehog combinatorial genelibrary can be cloned into the phagemid adjacent to the gIII signalsequence such that it will be expressed as a gIII fusion protein. Afterligation, the phagemid is used to transform competent E. coli TG1 cells.Transformed cells are subsequently infected with M13KO7 helper phage torescue the phagemid and its candidate hedgehog gene insert. Theresulting recombinant phage contain phagemid DNA encoding a specificcandidate hedgehog, and display one or more copies of the correspondingfusion coat protein. The phage-displayed candidate hedgehog proteinswhich are capable of binding an hedgehog receptor are selected orenriched by panning. For instance, the phage library can be applied tocells which express the patched protein and unbound phage washed awayfrom the cells. The bound phage is then isolated, and if the recombinantphage express at least one copy of the wild type gill coat protein, theywill retain their ability to infect E. coli. Thus, successive rounds ofreinfection of E. coli, and panning will greatly enrich for hedgehoghomologs, which can then be screened for further biological activitiesin order to differentiate agonists and antagonists.

Combinatorial mutagenesis has a potential to generate very largelibraries of mutant proteins, e.g., in the order of 10²⁶ molecules.Combinatorial libraries of this size may be technically challenging toscreen even with high throughput screening assays such as phage display.To overcome this problem, a new technique has been developed recently,recursive ensemble mutagenesis (REM), which allows one to avoid the veryhigh proportion of non-functional proteins in a random library andsimply enhances the frequency of functional proteins, thus decreasingthe complexity required to achieve a useful sampling of sequence space.REM is an algorithm which enhances the frequency of functional mutantsin a library when an appropriate selection or screening method isemployed (Arkin and Yourvan, 1992, PNAS USA 89:7811-7815; Yourvan etal., 1992, Parallel Problem Solving from Nature, 2., In Maenner andManderick, eds., Elsevir Publishing Co., Amsterdam, pp. 401-410;Delgrave et al., 1993, Protein Engineering 6(3):327-331).

The invention also provides for reduction of the hedgehog protein togenerate mimetics, e.g. peptide or non-peptide agents, which are able todisrupt binding of a hedgehog polypeptide of the present invention withan hedgehog receptor. Thus, such mutagenic techniques as described aboveare also useful to map the determinants of the hedgehog proteins whichparticipate in protein—protein interactions involved in, for example,binding of the subject hedgehog polypeptide to other extracellularmatrix components. To illustrate, the critical residues of a subjecthedgehog polypeptide which are involved in molecular recognition of anhedgehog receptor such as patched can be determined and used to generatehedgehog-derived peptidomimetics which competitively inhibit binding ofthe authentic hedgehog protein with that moiety. By employing, forexample, scanning mutagenesis to map the amino acid residues of each ofthe subject hedgehog proteins which are involved in binding otherextracellular proteins, peptidomimetic compounds can be generated whichmimic those residues of the hedgehog protein which facilitate theinteraction. Such mimetics may then be used to interfere with the normalfunction of a hedgehog protein. For instance, non-hydrolyzable peptideanalogs of such residues can be venerated using benzodiazepine (e.g.,see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., seeHuffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactamrings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylenepseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson etal. in Peptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), β-turndipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1:1231), and β-aminoalcohols(Gordon et al. (1985) Biochem Biophys Res Commun126:419; and Dann et al.(1986) Biochem Biophys Res Commun 134:71).

Recombinantly produced forms of the hedgehog proteins can be producedusing, e.g, expression vectors containing a nucleic acid encoding ahedgehog polypeptide, operably linked to at least one transcriptionalregulatory sequence. Operably linked is intended to mean that thenucleotide sequence is linked to a regulatory sequence in a manner whichallows expression of the nucleotide sequence. Regulatory sequences areart-recognized and are selected to direct expression of a hedgehogpolypeptide. Accordingly, the term transcriptional regulatory sequenceincludes promoters, enhancers and other expression control elements.Such regulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press. San Diego, Calif.(1990). For instance, any of a wide variety of expression controlsequences, sequences that control the expression of a DNA sequence whenoperatively linked to it, may be used in these vectors to express DNAsequences encoding hedgehog polypeptide. Such useful expression controlsequences, include, for example, a viral LTR, such as the LTR of theMoloney murine leukemia virus, the early and late promoters of SV40,adenovirus or cytomegalovirus immediate early promoter, the lac system,the trp system, the TAC or TRC system, T7 promoter whose expression isdirected by T7 RNA polymerase, the major operator and promoter regionsof phage λ, the control regions for fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g. Pho5, the promoters of the yeast α-matingfactors, the polyhedron promoter of the baculovirus system and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed and/orthe type of protein desired to be expressed. Moreover, the vector's copynumber, the ability to control that copy number and the expression ofany other proteins encoded by the vector, such as antibiotic markers,should also be considered.

In addition to providing a ready source of hedgehog polypeptides forpurification, the gene constructs of the present invention can also beused as a part of a gene therapy protocol to deliver nucleic acidsencoding either an agonistic or antagonistic form of a hedgehogpolypeptide. Thus, another aspect of the invention features expressionvectors for in vivo transfection of a hedgehog polypeptide in particularcell types so as cause ectopic expression of a hedgehog polypeptide inan epithelial tissue.

Formulations of such expression constructs may be administered in anybiologically effective carrier, e.g. any formulation or compositioncapable of effectively delivering the recombinant gene to cells in vivo.Approaches include insertion of the hedgehog coding sequence in viralvectors including recombinant retroviruses, adenovirus, adeno-associatedvirus, and herpes simplex virus-1, or recombinant bacterial oreukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNAcan be delivered with the help of, for example, cationic liposomes(lipofectin) or derivatized (e.g. antibody conjugated), polylysineconjugates, gramacidin S, artificial viral envelopes or other suchintracellular carriers, as well as direct injection of the geneconstruct or CaPO₄ precipitation carried out in vivo. It will beappreciated that because transduction of appropriate target cellsrepresents the critical first step in gene therapy, choice of theparticular gene delivery system will depend on such factors as thephenotype of the intended target and the route of administration, e.g.locally or systemically. Furthermore, it will be recognized that theparticular gene construct provided for in vivo transduction of hedgehogexpression are also useful for in vitro transduction of cells, such asfor use in the ex vivo tissue culture systems described below.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid, e.g. a cDNA,encoding the particular form of the hedgehog polypeptide desired.Infection of cells with a viral vector has the advantage that a largeproportion of the targeted cells can receive the nucleic acid.Additionally, molecules encoded within the viral vector, e.g., by a cDNAcontained in the viral vector, are expressed efficiently in cells whichhave taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery system of choice for thetransfer of exogenous genes in vivo, particularly into humans. Thesevectors provide efficient delivery of genes into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. A major prerequisite for the use of retroviruses is toensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment of specialized cell lines (termed “packaging cells”) whichproduce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arewell characterized for use in gene transfer for gene therapy purposes(for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid encoding ahedgehog polypeptide and renders the retrovirus replication defective.The replication defective retrovirus is then packaged into virions whichcan be used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals. Examples of suitable retrovirusesinclude pLJ, pZIP, pWE and pEM which are well known to those skilled inthe art. Examples of suitable packaging virus lines for preparing bothecotropic and amphotropic retroviral systems include Crip, Cre, 2 andAm. Retroviruses have been used to introduce a variety of genes intomany different cell types, including epithelial cells, in vitro and/orin vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398;Danos and Mulligan (1988) Proc. Natl. Acad Sci. USA 85:6460-6464; Wilsonet al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al.(1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991)Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl.Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al.(1992) Proc. Natl. Acad Sci. USA 89:10892-10895; Hwu et al. (1993) J.Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No.4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCTApplication WO 89/05345; and PCT Application WO 92/07573).

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234 andWO94/06920). For instance, strategies for the modification of theinfection spectrum of retroviral vectors include: coupling antibodiesspecific for cell surface antigens to the viral env protein (Roux et al.(1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255;and Goud et al. (1983) Virology 163:251-254); or coupling cell surfacereceptor ligands to the viral env proteins (Neda et al. (1991) J BiolChem 266:14143-14146). Coupling can be in the form of the chemicalcross-linking with a protein or other variety (e.g. lactose to convertthe env protein to an asialoglycoprotein), as well as by generatingfusion proteins (e.g. single-chain antibody/env fusion proteins). Thistechnique, while useful to limit or otherwise direct the infection tocertain tissue types, can also be used to convert an ecotropic vector into an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by theuse of tissue- or cell-specific transcriptional regulatory sequenceswhich control expression of the hedgehog gene of the retroviral vector.

Another viral gene delivery system useful in the present method utilizesadenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See for example Berkner et al. (1988)BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; andRosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 d1324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled inthe art. Recombinant adenoviruses can be advantageous in certaincircumstances in that they can be used to infect a wide variety of celltypes, including epithelial cells (Rosenfeld et al. (1992) cited supra).Furthermore, the virus particle is relatively stable and amenable topurification and concentration, and as above, can be modified so as toaffect the spectrum of infectivity. Additionally, introduced adenoviralDNA (and foreign DNA contained therein) is not integrated into thegenome of a host cell but remains episomal, thereby avoiding potentialproblems that can occur as a result of insertional mutagenesis insituations where introduced DNA becomes integrated into the host genome(e.g., retroviral DNA). Moreover, the carrying capacity of theadenoviral genome for foreign DNA is large (up to 8 kilobases) relativeto other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmandand Graham (1986) J. Virol. 57:267). Most replication-defectiveadenoviral vectors currently in use and therefore favored by the presentinvention are deleted for all or parts of the viral E1 and E3 genes butretain as much as 80% of the adenoviral genetic material (see, e.g.,Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham etal. in Methods in Molecular Biology, E. J. Murray, Ed. (Humana, Clifton,N.J., 1991) vol. 7. pp. 109-127). Expression of the inserted hedgehoggene can be under control of, for example, the E1A promoter, the majorlate promoter (MLP) and associated leader sequences, the E3 promoter, orexogenously added promoter sequences.

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of a hedgehogpolypeptide in the tissue of an animal. Most nonviral methods of genetransfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the hedgehog polypeptidegene by the targeted cell. Exemplary gene delivery systems of this typeinclude liposomal derived systems, poly-lysine conjugates, andartificial viral envelopes.

In clinical settings, the gene delivery systems for the therapeutichedgehog gene can be introduced into a patient by any of a number ofmethods, each of which is familiar in the art. For instance, apharmaceutical preparation of the gene delivery system can be introducedsystemically, e.g. by intravenous injection, and specific transductionof the protein in the target cells occurs predominantly from specificityof transfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994) PNAS 91: 3054-3057). A hedgehog expression construct can bedelivered in a gene therapy construct to dermal cells by, e.g.,electroporation using techniques described, for example, by Dev et al.((1994) Cancer Treat Rev 20:105-115).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

In yet another embodiment, the hedgehog or ptc therapeutic can be a“gene activation” construct which, by homologous recombination with agenomic DNA, alters the transcriptional regulatory sequences of anendogenous gene. For instance, the gene activation construct can replacethe endogenous promoter of a hedgehog gene with a heterologous promoter,e.g., one which causes consitutive expression of the hedgehog gene orwhich causes inducible expression of the gene under conditions differentfrom the normal expression pattern of the gene. Other genes in thepatched signaling pathway can be similarly targeted. A vareity ofdifferent formats for the gene activation constructs are available. See,for example, the Transkaryotic Therapies, Inc PCT publicationsWO93/09222, WO95/31560, WO96/29411, WO95/31560 and WO94/12650.

In preferred embodiments, the nucleotide sequence used as the geneactivation construct can be comprised of (1) DNA from some portion ofthe endogenous hedgehog gene (exon sequence, intron sequence, promotersequences, etc.) which direct recombination and (2) heterologoustranscriptional regulatory sequence(s) which is to be operably linked tothe coding sequence for the genomic hedgehog gene upon recombination ofthe gene activation construct. For use in generating cultures ofhedgehog producing cells, the construct may further include a reportergene to detect the presence of the knockout construct in the cell.

The gene activation construct is inserted into a cell, and integrateswith the genomic DNA of the cell in such a position so as to provide theheterologous regulatory sequences in operative association with thenative hedgehog gene. Such insertion occurs by homologous recombination,i.e., recombination regions of the activation construct that arehomologous to the endogenous hedgehog gene sequence hybridize to thegenomic DNA and recombine with the genomic sequences so that theconstruct is incorporated into the corresponding position of the genomicDNA.

The terms “recombination region” or “targeting sequence” refer to asegment (i.e., a portion) of a gene activation construct having asequence that is substantially identical to or substantiallycomplementary to a genomic gene sequence, e.g., including 5′ flankingsequences of the genomic gene, and can facilitate homologousrecombination between the genomic sequence and the targeting transgeneconstruct.

As used herein, the term “replacement region” refers to a portion of aactivation construct which becomes integrated into an endogenouschromosomal location following homologous recombination between arecombination region and a genomic sequence.

The heterologous regulatory sequences, e.g., which are provided in thereplacement region, can include one or more of a variety elements,including: promoters (such as constitutive or inducible promoters),enhancers, negative regulatory elements, locus control regions,transcription factor binding sites, or combinations thereof.Promoters/enhancers which may be used to control the expression of thetargeted gene in vivo include, but are not limited to, thecytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., 1989, J.Exp. Med., 169:13), the human β-actin promoter (Gunning et al. (1987)PNAS 84:4831-4835), the glucocorticoid-inducible promoter present in themouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig etal. (1984) Mol. Cell Biol. 4:1354-1362), the long terminal repeatsequences of Moloney murine leukemia virus (MuLV LTR) (Weiss et al.(1985) RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.), the SV40 early or late region promoter (Bernoist et al.(1981) Nature 290:304-310; Templeton et al. (1984) Mol. Cell Biol.,4:817; and Sprague et al. (1983) J. Virol., 45:773), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (RSV)(Yamamoto et al., 1980, Cell, 22:787-797), the herpes simplex virus(IISV) thymidine kinase promoter/enhancer (Wagner et al. (1981) PNAS82:3567-71), and the herpes simplex virus LAT promoter (Wolfe et al.(1992) Nature Genetics, 1:379-384).

In an exemplary embodiment, portions of the 5′ flanking region of thehuman Shh gene are amplified using primers which add restriction sites,to generate the following fragments

5′-gcgcgcttcgaaGCGAGGCAGCCAGCGAGGGAGAGAGCGAGCGGGCGAGCCGGAGC-GAGGAAatcgatgcgcgc (primer 1)5′-gcgcgcagatctGGGAAAGCGCAAGAGAGAGCGCACACGCACACACCCGCCGCGCG-CACTCGggatccgcgcgc (primer 2)

As illustrated, primer 1 includes a 5′ non-coding region of the humanShh gene and is flanked by an AsuII and ClaI restriction sites. Primer 2includes a portion of the 5′ non-coding region immediately 3′ to thatpresent in primer 1. The hedgehog gene sequence is flanked by XhoII andBamHI restriction sites. The purified amplimers are cut with each of theenzymes as appropriate.

The vector pCDNA1.1 (Invitrogen) includes a CMV promoter. The plasmid iscut with with AsuII, which cleaves just 3′ to the CMV promoter sequence.The AsuII/ClaI fragment of primer 1 is ligated to the AsuII cleavagesite of the pcDNA vector. The ClaI/AsuII ligation destroys the AsuIIsite at the 3′ end of a properly inserted primer 1.

The vector is then cut with BamHI, and an XhoII/BamHI fragment of primer2 is ligated to the BamHI cleavage site. As above, the BamHI/XhoIIligation destroys the BamHI site at the 5′ end of a properly insertedprimer 2.

Individual colonies are selected, cut with AsuII and BamHI, and the sizeof the AsuII/BamHII fragment determined. Colonies in which both theprimer 1 and primer 2 sequences are correctly inserted are furtheramplified, an cut with AsuII and BamHI to produce the gene activationconstruct

cgaagcgaggcagccagcgagggagagagcgagcgggcgagccggagcgaggaaATCGAAGGTTCGAATCCTTCCCCCACCACCATCACTTTCAAAAGTCCGAAAGAATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGTAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTTGGTACCGAGCTCGGATCgatctgggaaagcgcaagagagagcgcacacgcacacacccgccgcgcgcactcgg

In this construct, the flanking primer 1 and primer 2 sequences providethe recombination region which permits the insertion of the CMV promoterin front of the coding sequence for the human Shh gene. Otherheterologous promoters (or other transcriptional regulatory sequences)can be inserted in a genomic hedgehog gene by a similar method.

In still other embodiments, the replacement region merely deletes anegative transcriptional control element of the native gene, e.g., toactivate expression, or ablates a positive control element, e.g., toinhibit expression of the targeted gene.

V. Exemplary ptc Therapeutic Compounds

In another embodiment, the subject method is carried out using a ptctherapeutic composition. Such compositions can be generated with, forexample, compounds which bind to patched and alter its signaltransduction activity, compounds which alter the binding and/orenzymatic activity of a protein (e.g., intracellular) involved inpatched signal pathway, and compounds which alter the level ofexpression of a hedgehog protein, a patched protein or a proteininvolved in the intracellular signal transduction pathway of patched.

The availability of purified and recombinant hedgehog polypeptidesfacilitates the generation of assay systems which can be used to screenfor drugs, such as small organic molecules, which are either agonists orantagonists of the normal cellular function of a hedgehog and/or patchedprotein, particularly their role in the pathogenesis of epithelial cellproliferation and/or differentiation. In one embodiment, the assayevaluates the ability of a compound to modulate binding between ahedgehog polypeptide and a hedgehog receptor such as patched. In otherembodiments, the assay merely scores for the ability of a test compoundto alter the signal transduction acitity of the patched protein. In thismanner, a variety of hedgehog and/or ptc therapeutics, bothproliferative and anti-proliferative in activity, can be identified. Avariety of assay formats will suffice and, in light of the presentdisclosure, will be comprehended by skilled artisan.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with receptorproteins.

Acordingly, in an exemplary screening assay for ptc therapeutics, thecompound of interest is contacted with a mixture including a hedgehogreceptor protein (e.g., a cell expressing the patched receptor) and ahedgehog protein under conditions in which it is ordinarily capable ofbinding the hedgehog protein. To the mixture is then added a compositioncontaining a test compound. Detection and quantification ofreceptor/hedgehog complexes provides a means for determining the testcompound's efficacy at inhibiting (or potentiating) complex formationbetween the receptor protein and the hedgehog polypeptide. The efficacyof the compound can be assessed by generating dose response curves fromdata obtained using various concentrations of the test compound.Moreover, a control assay can also be performed to provide a baselinefor comparison. In the control assay, isolated and purified hedgehogpolypeptide is added to the receptor protein, and the formation ofreceptor/hedgehog complex is quantitated in the absence of the testcompound.

In other embodiments, a ptc therapeutic of the present invention is onewhich disrupts the association of patched with smoothened.

Agonist and antagonists of epithelial cell growth can be distinguished,and the efficacy of the compound can be assessed, by subsequent testingwith epithelial cells, e.g., in culture.

In an illustrative embodiment, the polypeptide utilized as a hedgehogreceptor can be generated from the patched protein. Accordingly, anexemplary screening assay includes all or a suitable portion of thepatched protein which can be obtained from, for example, the humanpatched gene (GenBank U43148) or other vertebrate sources (see GenBankAccession numbers U40074 for chicken patched and U46155 for mousepatched), as well as from drosophila (GenBank Accession number M28999)or other invertebrate sources. The patched protein can be provided inthe screening assay as a whole protein (preferably expressed on thesurface of a cell), or alternatively as a fragment of the full lengthprotein which binds to hedgehog polypeptides, e.g., as one or both ofthe substantial extracellular domains (e.g. corresponding to residuesAsn120-Ser438 and/or Arg770-Trp1027 of the human patched protein—whichare also potential antagonists of hedgehog-dependent signaltransduction). For instance, the patched protein can be provided insoluble form, as for example a preparation of one of the extracellulardomains, or a preparation of both of the extracellular domains which arecovalently connected by an unstructured linker (see, for example, Hustonet al. (1988) PNAS 85:4879; and U.S. Pat. No. 5,091,513). In otherembodiments, the protein can be provided as part of a liposomalpreparation or expressed on the surface of a cell. The patched proteincan derived from a recombinant gene, e.g., being ectopically expressedin a heterologous cell. For instance, the protein can be expressed onoocytes, mammalian cells (e.g., COS, CHO, 3T3 or the like), or yeastcell by standard recombinant DNA techniques. These recombinant cells canbe used for receptor binding, signal transduction or gene expressionassays. Marigo et al. (1996) Development 122:1225-1233 illustrates abinding assay of human hedgehog to chick patched protein ectopicallyexpressed in Xenopus laevis oocytes. The assay system of Marigo et al.can be adapted to the present drug screening assays. As illustrated inthat reference, Shh binds to the patched protein in a selective,saturable, dose-dependent manner, thus demonstrating that patched is areceptor for Shh.

Complex formation between the hedgehog polypeptide and a hedgehogreceptor may be detected by a variety of techniques. For instance,modulation of the formation of complexes can be quantitated using, forexample, detectably labelled proteins such as radiolabelled,fluorescently labelled, or enzymatically labelled hedgehog polypeptides,by immunoassay, or by chromatographic detection.

Typically, for cell-free assays, it will be desirable to immobilizeeither the hedgehog receptor or the hedgehog polypeptide to facilitateseparation of receptor/hedgehog complexes from uncomplexed forms of oneof the proteins, as well as to accommodate automation of the assay. Inone embodiment, a fusion protein can be provided which adds a domainthat allows the protein to be bound to a matrix. For example,glutathione-S-transferase/receptor (GST/receptor) fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical. St. Louis.Mo.) or glutathione derivatized microtitre plates, which are thencombined with the hedgehog polypeptide, e.g. an ³⁵S-labeled hedgehogpolypeptide, and the test compound and incubated under conditionsconducive to complex formation, e.g. at physiological conditions forsalt and pH, though slightly more stringent conditions may be desired.Following incubation, the beads are washed to remove any unboundhedgehog polypeptide, and the matrix bead-bound radiolabel determineddirectly (e.g. beads placed in scintillant), or in the supernatant afterthe receptor/hedgehog complexes are dissociated. Alternatively, thecomplexes can be dissociated from the bead, separated by SDS-PAGE gel,and the level of hedgehog polypeptide found in the bead fractionquantitated from the gel using standard electrophoretic techniques.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, soluble portionsof the hedgehog receptor protein can be immobilized utilizingconjugation of biotin and streptavidin. For instance, biotinylatedreceptor molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with the hedgehog receptor but whichdo not interfere with hedgehog binding can be derivatized to the wellsof the plate, and the receptor trapped in the wells by antibodyconjugation. As above, preparations of a hedgehog polypeptide and a testcompound are incubated in the receptor-presenting wells of the plate,and the amount of receptorlhedgehog complex trapped in the well can bequantitated. Exemplary methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the hedgehogpolypeptide, or which are reactive with the receptor protein and competefor binding with the hedgehog polypeptide; as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thehedgehog polypeptide. In the instance of the latter, the enzyme can bechemically conjugated or provided as a fusion protein with the hedgehogpolypeptide. To illustrate, the hedgehog polypeptide can be chemicallycross-linked or genetically fused with alkaline phosphatase, and theamount of hedgehog polypeptide trapped in the complex can be assessedwith a chromogenic substrate of the enzyme, e.g.paranitrophenylphosphate. Likewise, a fusion protein comprising thehedgehog polypeptide and glutathione-S-transferase can be provided, andcomplex formation quantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:71 30).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the protein, such asthe anti-hedgehog antibodies described herein, can be used.Alternatively, the protein to be detected in the complex can be “epitopetagged” in the form of a fusion protein which includes, in addition tothe hedgehog polypeptide or hedgehog receptor sequence, a secondpolypeptide for which antibodies are readily available (e.g. fromcommercial sources). For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) whichincludes a 10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharamacia, N.J.).

Where the desired portion of the hedgehog receptor (or other hedgehogbinding molecule) cannot be provided in soluble form, liposomal vesiclescan be used to provide manipulatable and isolatable sources of thereceptor. For example, both authentic and recombinant forms of thepatched protein can be reconstituted in artificial lipid vesicles (e.g.phosphatidylcholine liposomes) or in cell membrane-derived vesicles(see, for example. Bear et al. (1992) Cell 68:809-818; Newton et al.(1983) Biochemistry 22:6110-6117; and Reber et al. (1987) J Biol Chem262:11369-11374).

In addition to cell-free assays, such as described above, the readilyavailable source of hedgehog proteins provided by the art alsofacilitates the generation of cell-based assays for identifying smallmolecule agonists/antagonists and the like. Analogous to the cell-basedassays described above for screening combinatorial libraries, cellswhich are sensitive to hedgehog induction, e.g. patched-expressing cellsor other epithelially-derived cells sensitive to hedgehog induction, canbe contacted with a hedgehog protein and a test agent of interest, withthe assay scoring for anything from simple binding to the cell tomodulation in hedgehog inductive responses by the target cell in thepresence and absence of the test agent. As with the cell-free assays,agents which produce a statistically significant change in hedgehogactivities (either inhibition or potentiation) can be identified.

In other emdodiments, the cell-based assay scores for agents whichdisrupt association of patched and smoothened proteins, e.g., in thecell surface membrane or liposomal preparation.

In addition to characterizing cells that naturally express the patchedprotein, cells which have been genetically engineered to ectopicallyexpress patched can be utilized for drug screening assays. As anexample, cells which either express low levels or lack expression of thepatched protein, e.g. Xenopus laevis oocytes, COS cells or yeast cells,can be genetically modified using standard techniques to ectopicallyexpress the patched protein. (see Marigo et al., supra).

The resulting recombinant cells, e.g., which express a functionalpatched receptor, can be utilized in receptor binding assays to identifyagonist or anatagonsts of hedgehog binding. Binding assays can beperformed using whole cells. Furthermore, the recombinant cells of thepresent invention can be engineered to include other heterolgous genesencoding proteins involved in hedgehog-dependent signal pathways. Forexample, the gene products of one or more of smoothened, costal-2 and/orfused can be co-expressed with patched in the reagent cell, with assaysbeing sensitive to the functional reconstituion of the hedgehog signaltransduction cascade.

Alternatively, liposomal preparations using reconstituted patchedprotein can be utilized. Patched protein purified from detergentextracts from both authentic and recombinant origins can bereconstituted in in artificial lipid vesicles (e.g. phosphatidylcholineliposomes) or in cell membrane-derived vesicles (see, for example, Bearet al. (1992) Cell 68:809-818; Newton et al. (1983) Biochemistry22:6110-6117; and Reber et al. (1987) J Biol Chem 262:11369-11374). Thelamellar structure and size of the resulting liposomes can becharacterized using electron microscopy. External orientation of thepatched protein in the reconstituted membranes can be demonstrated, forexample, by immunoelectron microscopy. The hedgehog protein bindingactivity of liposomes containing patched and liposomes without theprotein in the presence of candidate agents can be compared in order toidentify potential modulators of the hedgehog-patched interaction.

The hedgehog protein used in these cell-based assays can be provided asa purified source (natural or recombinant in origin), or in the form ofcells/tissue which express the protein and which are co-cultured withthe target cells. As in the cell-free assays, where simple binding(rather than induction) is the hedgehog activity scored for in theassay, the protein can be labelled by any of the above-mentionedtechniques, e.g., fluorescently, enzymatically or radioactively, ordetected by immunoassay.

In addition to binding studies, functional assays can be used toidentified modulators, i.e., agonists or antagonists, of hedgehog orpatched activities. By detecting changes in intracellular signals, suchas alterations in second messengers or gene expression, inpatched-expressing cells contacted with a test agent, candidate agonistsand antagonists to patched signaling can be identified.

A number of gene products have been implicated in patched-mediatedsignal transduction, including patched, the transcription factor cubitusinterruptus (ci), the serine/threonine kinase fused (fu) and the geneproducts of costal-2, smoothened and suppressor of fused.

The interaction of a hedgehog protein with patched sets in motion acascade involving the activation and inhibition of downstream effectors,the ultimate consequence of which is, in some instances, a detectablechange in the transcription or translation of a gene. Potentialtranscriptional targets of patched signaling are the patched gene itself(Hidalgo and Ingham, 1990 Development 110, 291-301; Marigo et al., 1996)and the vertebrate homologs of the drosophila cubitus interruptus gene,the GLI genes (Hui et al. (1994) Dev Biol 162:402-413). Patched geneexpression has been shown to be induced in cells of the limb bud and theneural plate that are responsive to Shh. (Marigo et al. (1996) PNAS, inpress; Marigo et al. (1996) Development 122:1225-1233). The GLI genesencode putative transcription factors having zinc finger DNA bindingdomains (Orenic et al. (1990) Genes & Dev 4:1053-1067; Kinzler et al.(1990), Mol Cell Biol 10:634-642). Transcription of the GLI gene hasbeen reported to be upregulated in response to hedgehog in limb buds,while transcription of the GLI3 gene is downregulated in response tohedgehog induction (Marigo et al. (1996) Development 122:1225-1233). Byselecting transcriptional regulatory sequences from such target genes,e.g. from patched or GLI genes, that are responsible for the up- or downregulation of these genes in response to patched signalling, andoperatively linking such promoters to a reporter gene, one can derive atranscription based assay which is sensitive to the ability of aspecific test compound to modify patched signalling pathways. Expressionof the reporter gene, thus, provides a valuable screening tool for thedevelopment of compounds that act as agonists or antagonists of ptcinduction of differentiation/quiescence.

Reporter gene based assays of this invention measure the end stage ofthe above described cascade of events, e.g., transcriptional modulation.Accordingly, in practicing one embodiment of the assay, a reporter geneconstruct is inserted into the reagent cell in order to generate adetection signal dependent on ptc signaling. To identify potentialregulatory elements responsive to ptc signaling present in thetranscriptional regulatory sequence of a target gene, nested deletionsof genomic clones of the target gene can be constructed using standardtechniques. See, for example, Current Protocols in Molecular Biology,Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989); U.S.Pat. No. 5,266,488; Sato et al. (1995) J Biol Chem 270:10314-10322; andKube et al. (1995) Cytokine 7:1-7. A nested set of DNA fragments fromthe gene's 5′-flanking region are placed upstream of a reporter gene,such as the luciferase gene, and assayed for their ability to directreporter gene expression in patched expressing cells. Host cellstransiently transfected with reporter gene constructs can be scored forthe induction of expression of the reporter gene in the presence andabsence of hedgehog to determine regulatory sequences which areresponsice to patched-dependent signalling.

In practicing one embodiment of the assay, a reporter gene construct isinserted into the reagent cell in order to generate a detection signaldependent on second messengers generated by induction with hedgehogprotein. Typically, the reporter gene construct will include a reportergene in operative linkage with one or more transcriptional regulatoryelements responsive to the hedgehog activity, with the level ofexpression of the reporter gene providing the hedgehog-dependentdetection signal. The amount of transcription from the reporter gene maybe measured using any method known to those of skill in the art to besuitable. For example, mRNA expression from the reporter gene may bedetected using RNAse protection or RNA-based PCR, or the protein productof the reporter gene may be identified by a characteristic stain or anintrinsic activity. The amount of expression from the reporter gene isthen compared to the amount of expression in either the same cell in theabsence of the test compound (or hedgehog) or it may be compared withthe amount of transcription in a substantially identical cell that lacksthe target receptor protein. Any statistically or otherwise significantdifference in the amount of transcription indicates that the testcompound has in some manner altered the signal transduction of thepatched protein, e.g., the test compound is a potential ptc therapeutic.

As described in further detail below, in preferred embodiments the geneproduct of the reporter is detected by an intrinsic activity associatedwith that product. For instance, the reporter gene may encode a geneproduct that, by enzymatic activity, gives rise to a detection signalbased on color, fluorescence, or luminescence. In other preferredembodiments, the reporter or marker gene provides a selective growthadvantage, e.g., the reporter gene may enhance cell viability, relieve acell nutritional requirement, and/or provide resistance to a drug.

Preferred reporter genes are those that are readily detectable. Thereporter gene may also be included in the construct in the form of afusion gene with a gene that includes desired transcriptional regulatorysequences or exhibits other desirable properties. Examples of reportergenes include, but are not limited to CAT (chloramphenicol acetyltransferase) (Alton and Vapnek (1979). Nature 282: 864-869) luciferase,and other enzyme detection systems, such as beta-galactosidase; fireflyluciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterialluciferase (Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwinet al. (1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh etal. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol.Appl. Gen. 2: 101), human placental secreted alkaline phosphatase(Cullen and Malim (1992) Methods in Enzymol. 216:362-368).

Transcriptional control elements which may be included in a reportergene construct include, but are not limited to, promoters, enhancers,and repressor and activator binding sites. Suitable transcriptionalregulatory elements may be derived from the transcriptional regulatoryregions of genes whose expression is induced after modulation of apatched signal transduction pathway. The characteristics of preferredgenes from which the transcriptional control elements are derivedinclude, but are not limited to, low or undetectable expression inquiescent cells, rapid induction at the transcriptional level withinminutes of extracellular simulation, induction that is transient andindependent of new protein synthesis, subsequent shut-off oftranscription requires new protein synthesis, and mRNAs transcribed fromthese genes have a short half-life. It is not necessary for all of theseproperties to be present.

In yet other embodiments, second messenger generation can be measureddirectly in the detection step, such as mobilization of intracellularcalcium, phospholipid metabolism or adenylate cyclase activity arequantitated, for instance, the products of phospholipid hydrolysis IP₃.DAG or cAMP could be measured For example, recent studies haveimplicated protein kinase A (PKA) as a possible component ofhedgehog/patched signaling (Hammerschmidt et al. (1996) Genes & Dev10:647). High PKA activity has been shown to antagonize hedgehogsignaling in these systems. Although it is unclear whether PKA actsdirectly downstream or in parallel with hedgehog signaling, it ispossible that hedgehog signalling occurs via inhibition of PKA activity.Thus, detection of PKA activity provides a potential readout for theinstant assays.

In a preferred embodiment, the ptc therapeutic is a PKA inhibitor. Avariety of PKA inhibitors arc known in the art, including both peptidyland organic compounds. For instance, the ptc therapeutic can be a5-isoquinolinesulfonamide, such as represented in the general formula:

wherein,

R₁ and R₂ each can independently represent hydrogen, and as valence andstability permit a lower alkyl, a lower alkenyl, a lower alkynyl, acarbonyl (such as a carboxyl, an ester, a formate, or a ketone), athiocarbonyl (such as a thioester, a thioacetate, or a thioformate), anamino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, asulfonate, a sulfonamido, —(CH₂)_(m)—R₈, —(CH2)_(m)—OH,—(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₈, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₈, or

R₁ and R₂ taken together with N form a heterocycle (substituted orunsubstituted);

R₃ is absent or represents one or more substitutions to the isoquinolinering such as a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl(such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl(such as a thioester, a thioacetate, or a thioformate), an amino, anacylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate,a sulfonamido, —(CH₂)_(m)—R₈, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₈, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₈;

R₈ represents a substituted or unsubstituted aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle, and

n and m are independently for each occurrence zero or an integer in therange of 1 to 6.

In a preferred embodiment, the PKA inhibitor isN-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89;Calbiochem Cat. No. 371963), e.g., having the formula:

In another embodiment, the TKA inhibitor is1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7; Calbiochem Cat. No.371955), e.g., having the formula:

In still other embodiments, the PKA inhibitor is KT5720 (Calbiochem Cat.No. 420315), having the structure

A variety of nucleoside analogs are also useful as PKA inhibitors. Forexample, the subject method can be carried out cyclic AMP analogs whichinhibit the kinase activity of PKA, as for example, 8-bromo-cAMP ordibutyryl-cAMP

Exemplary peptidyl inhibitors of PKA activity include the PKA HeatStable Inhibitor (isoform α; see, for example, Calbiochem Cat. No.539488, and Wen et al. (1995) J Biol Chem 270:2041).

Certain hedgehog receptors may stimulate the activity of phospholipases.Inositol lipids can be extracted and analyzed using standard lipidextraction techniques. Water soluble derivatives of all three inositollipids (IP₁, IP₂, IP₃) can also be quantitated using radiolabellingtechniques or HPLC.

The mobilization of intracellular calcium or the influx of calcium fromoutside the cell may be a response to hedgehog stimulation or lack thereof. Calcium flux in the reagent cell can be measured using standardtechniques. The choice of the appropriate calcium indicator,fluorescent, bioluminescent, metallochromic, or Ca⁺⁺-sensitivemicroelectrodes depends on the cell type and the magnitude and timeconstant of the event under study (Borle (1990) Environ Health Perspect84:45-56). As an exemplary method of Ca⁺⁺ detection, cells could beloaded with the Ca⁺⁺ sensitive fluorescent dye fura-2 or indo-1, usingstandard methods, and any change in Ca⁺⁺ measured using a fluorometer.

In certain embodiments of the assay, it may be desirable to screen forchanges in cellular phosphorylation. As an example, the drosophila genefused (fu) which encodes a serine/threonine kinase has been identifiedas a potential downstream target in hedgehog signaling. (Preat et al.,1990 Nature 347, 87-89; Therond et al. 1993, Mech. Dev. 44. 65-80). Theability of compounds to modulate serine/threonine kinase activationcould be screened using colony immunoblotting (Lyons and Nelson (1984)Proc. Natl. Acad. Sci. USA 81:7426-7430) using antibodies againstphosphorylated serine or threonine residues. Reagents for performingsuch assays are commercially available, for example, phosphoserine andphosphothreonine specific antibodies which measure increases inphosphorylation of those residues can be purchased from commercialsources.

In yet another embodiment, the ptc therapeutic is an antisense moleculewhich inhibits expression of a protein involved in a patched-mediatedsignal transduction pathway. To illustrate, by inhibiting the expressionof a protein which are involved in patched signals, such as fused,costal-2, smoothened and/or Gli genes, the ability of the patched signalpathway(s) to inhibit proliferation of a cell can be altered, e.g.,potentiated or repressed.

As used herein, “antisense” therapy refers to administration or in situgeneration of oligonucleotide probes or their derivatives whichspecifically hybridize (e.g. bind) under cellular conditions withcellular mRNA and/or genomic DNA encoding a hedgehog protein, patched,or a protein involved in patched-mediated signal transduction. Thehybridization should inhibit expression of that protein, e.g. byinhibiting transcription and/or translation. The binding may be byconventional base pair complementarity, or, for example, in the case ofbinding to DNA duplexes, through specific interactions in the majorgroove of the double helix. In general, “antisense” therapy refers tothe range of techniques generally employed in the art, and includes anytherapy which relies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thetarget cellular mRNA. Alternatively, the antisense construct is anoligonucleotide probe which is generated ex vivo and which, whenintroduced into the cell causes inhibition of expression by hybridizingwith the mRNA and/or genomic sequences of a target gene. Sucholigonucleotide probes are preferably modified oligonucleotide which areresistant to endogenous nucleases, e.g. exonucleases and/orendonucleases, and is therefore stable in vivo. Exemplary nucleic acidmolecules for use as antisense oligonucleotides are phosphoramidate,phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat.Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, generalapproaches to constructing oligomers useful in antisense therapy havebeen reviewed, for example, by Van der Krol et al. (1988) Biotechniques6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668.

Several considerations should be taken into account when constructingantisense oligonucleotides for the use in the methods of the invention:(1) oligos should have a GC content of 50% or more; (2) avoid sequenceswith stretches of 3 or more G's; and (3) oligonucleotides should not belonger than 25-26 mers. When testing an antisense oligonucleotide, amismatched control can be constructed. The controls can be generated byreversing the sequence order of the corresponding antisenseoligonucleotide in order to conserve the same ratio of bases.

In an illustrative embodiment, the ptc therapeutic can be an antisenseconstruct for inhibiting the expression of patched, e.g., to mimic theinhibition of patched by hedgehog. Exemplary antisense constructsinclude:

5′-GTCCTGGCGCCGCCGCCGCCGTCGCC 5′-TTCCGATGACCGGCCTTTCGCGGTGA5′-GTGCACGGAAAGGTGCAGGCCACACT

VI. Exemplary Pharmaceutical Preparations of Hedgehog and ptcTherapeutics

The source of the hedgehog and ptc therapeutics to be formulated willdepend on the particular form of the agent. Small organic molecules andpeptidyl fragments can be chemically synthesized and provided in a pureform suitable for pharmaceutical/cosmetic usage. Products of naturalextracts can be purified according to techniques known in the art. Forexample, the Cox et al. U.S. Pat. No. 5,286,654 describes a method forpurifying naturally occurring forms of a secreted protein and can beadapted for purification of hedgehog polypeptides. Recombinant sourcesof hedgehog polypeptides are also available. For example, the geneencoding hedgehog polypeptides, are known, inter alia, from PCTpublications WO 95/18856 and WO 96/17924.

Those of skill in treating epithelial tissues can determine theeffective amount of an hedgehog or ptc therapeutic to be formulated in apharmaceutical or cosmetic preparation.

The hedgehog or ptc therapeutic formulations used in the method of theinvention are most preferably applied in the form of appropriatecompositions. As appropriate compositions there may be cited allcompositions usually employed for systemically or topicallyadministering drugs. The pharmaceutically acceptable carrier should besubstantially inert, so as not to act with the active component.Suitable inert carriers include water, alcohol polyethylene glycol,mineral oil or petroleum gel, propylene glycol and the like.

To prepare the pharmaceutical compositions of this invention, aneffective amount of the particular hedgehog or ptc therapeutic as theactive ingredient is combined in intimate admixture with apharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirable inunitary dosage form suitable, particularly, for administration orally,rectally, percutaneously, or by parenteral injection. For example, inpreparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed such as, for example, water,glycols, oils, alcohols and the like in the case of oral liquidpreparations such as suspensions, syrups, elixirs and solutions; orsolid carriers such as starches, sugars, kaolin, lubricants, binders,disintegrating agents and the like in the case of powders, pills,capsules, and tablets. Because of their ease in administration, tabletsand capsules represents the most advantageous oral dosage unit form, inwhich case solid pharmaceutical carriers are obviously employed. Forparenteral compositions, the carrier will usually comprise sterilewater, at least in large part, though other ingredients, for example, toaid solubility, may be included. Injectable solutions, for example, maybe prepared in which the carrier comprises saline solution, glucosesolution or a mixture of saline and glucose solution. Injectablesuspensions may also be prepared in which case appropriate liquidcarriers, suspending agents and the like may be employed. Also includedare solid form preparations which are intended to be converted, shortlybefore use, to liquid form preparations. In the compositions suitablefor percutaneous administration, the carrier optionally comprises apenetration enhancing agent and/or a suitable wetting agent, optionallycombined with suitable additives of any nature in minor proportions,which additives do not introduce a significant deleterious effect on theskin.

In addition to the direct topical application of the preparations theycan be topically administered by other methods, for example,encapsulated in a temperature and/or pressure sensitive matrix or infilm or solid carrier which is soluble in body fluids and the like forsubsequent release, preferably sustained-release of the activecomponent.

As appropriate compositions for topical application there may be citedall compositions usually employed for topically administeringtherapeutics, e.g., creams, gellies, dressings, shampoos, tinctures,pastes, ointments, salves, powders, liquid or semiliquid formulation andthe like. Application of said compositions may be by aerosol e.g. with apropellent such as nitrogen carbon dioxide, a freon, or without apropellent such as a pump spray, drops, lotions, or a semisolid such asa thickened composition which can be applied by a swab. In particularcompositions, semisolid compositions such as salves, creams, pastes,gellies, ointments and the like will conveniently be used.

It is especially advantageous to formulate the subject compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used in the specification and claims herein refersto physically discreate units suitable as unitary dosages, each unitcontaining a predetermined quantity of active ingredient calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. Examples of such dosage unit forms are tablets(including scored or coated tablets), capsules, pills, powders packets,wafers, injectable solutions or suspensions, teaspoonfuls,tablespoonfuls and the like, and segregated multiples thereof.

The pharmaceutical preparations of the present invention can be used, asstated above, for the many applications which can be considered cosmeticuses. Cosmetic compositions known in the art, preferably hypoallergicand pH controlled are especially preferred, and include toilet waters,packs, lotions, skin milks or milky lotions. The preparations contain,besides the hedgehog or ptc therapeutic, components usually employed insuch preparations. Examples of such components are oils, fats, waxes,surfactants, humectants, thickening agents, antioxidants, viscositystabilizers, chelating agents, buffers, preservatives, perfumes,dyestuffs, lower alkanols, and the like. If desired, further ingredientsmay be incorporated in the compositions, e.g. antiinflammatory agents,antibacterials, antifungals, disinfectants, vitamins, sunscreens,antibiotics, or other anti-acne agents.

Examples of oils comprise fats and oils such as olive oil andhydrogenated oils; waxes such as beeswax and lanolin; hydrocarbons suchas liquid paraffin, ceresin, and squalane; fatty acids such as stearicacid and oleic acid; alcohols such as cetyl alcohol, stearyl alcohol,lanolin alcohol, and hexadecanol; and esters such as isopropylmyristate, isopropyl palmitate and butyl stearate. As examples ofsurfactants there may be cited anionic surfactants such as sodiumstearate, sodium cetylsulfate, polyoxyethylene laurylether phosphate,sodium N-acyl glutamate; cationic surfactants such asstearyldimethylbenzylammonium chloride and stearyltrimethylammoniumchloride; ampholytic surfactants such as alkylaminoethylglycinehydrocloride solutions and lecithin; and nonionic surfactants such asglycerin monostearate, sorbitan monostearate, sucrose fatty acid esters,propylene glycol monostearate, polyoxyethylene oleylether, polyethyleneglycol monostearate, polyoxyethylene sorbitan monopalmitate,polyoxyethylene coconut fatty acid monoethanolamide, polyoxypropyleneglycol (e.g. the materials sold under the trademark “Pluronic”),polyoxyethylene castor oil, and polyoxyethylene lanolin. Examples ofhumectants include glycerin, 1,3-butylene glycol, and propylene glycol;examples of lower alcohols include ethanol and isopropanol; examples ofthickening agents include xanthan gum, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, polyethylene glycol and sodiumcarboxymethyl cellulose; examples of antioxidants comprise butylatedhydroxytoluene, butylated hydroxyanisole, propyl gallate, citric acidand ethoxyquin; examples of chelating agents include disodium edetateand ethanehydroxy diphosphate; examples of buffers comprise citric acid,sodium citrate, boric acid, borax, and disodium hydrogen phosphate; andexamples of preservatives are methyl parahydroxybenzoate, ethylparahydroxybenzoate, dehydroacetic acid, salicylic acid and benzoicacid.

For preparing ointments, creams, toilet waters, skin milks, and thelike, typically from 0.01 to 10% in particular from 0.1 to 5% and morein particular from 0.2 to 2.5% of the active ingredient, e.g., of thehedgehog or ptc therapeutic, will be incorporated in the compositions.In ointments or creams, the carrier for example consists of 1 to 20%, inparticular 5 to 15% of a humectant, 0.1 to 10% in particular from 0.5 to5% of a thickener and water; or said carrier may consist of 70 to 99%,in particular 20 to 95% of a surfactant, and 0 to 20%, in particular 2.5to 15% of a fat; or 80 to 99.9% in particular 90 to 99% of a thickener;or 5 to 15% of a surfactant, 2-15% of a humectant, 0 to 80% of an oil,very small (<2%) amounts of preservative, coloring agent and/or perfume,and water. In a toilet water, the carrier for example consists of 2 to10% of a lower alcohol, 0.1 to 10% or in particular 0.5 to 1% of asurfactant, 1 to 20%, in particular 3 to 7% of a humectant, 0 to 5% of abuffer, water and small amounts (<2%) of preservative, dyestuff and/orperfume. In a skin milk, the carrier typically consists of 10-50% ofoil, 1 to 10% of surfactant, 50-80% of water and 0 to 3% of preservativeand/or perfume. In the aforementioned preparations, all % symbols referto weight by weight percentage.

Particular compositions for use in the method of the present inventionare those wherein the hedgehog or ptc therapeutic is formulated inliposome-containing compositions. Liposomes are artificial vesiclesformed by amphiphatic molecules such as polar lipids, for example,phosphatidyl cholines, ethanolamines and serines, sphingomyelins,cardiolipins, plasmalogens, phosphatidic acids and cerebiosides.Liposomes are formed when suitable amphiphathic molecules are allowed toswell in water or aqueous solutions to form liquid crystals usually ofmultilayer structure comprised of many bilayers separated from eachother by aqueous material (also referred to as coarse liposomes).Another type of liposome known to be consisting of a single bilayerencapsulating aqueous material is referred to as a unilamellar vesicle.If water-soluble materials are included in the aqueous phase during theswelling of the lipids they become entrapped in the aqueous layerbetween the lipid bilayers.

Water-soluble active ingredients such as, for example, various saltforms of a hedgehog polypeptide, are encapsulated in the aqueous spacesbetween the molecular layers. The lipid soluble active ingredient ofhedgehog or ptc therapeutic, such as an organic mimetic, ispredominantly incorporated into the lipid layers, although polar headgroups may protrude from the layer into the aqueous space. Theencapsulation of these compounds can be achieved by a number of methods.The method most commonly used involves casting a thin film ofphospholipid onto the walls of a flask by evaporation from an organicsolvent. When this film is dispersed in a suitable aqueous mediummultilamellar liposomes are formed. Upon suitable sonication, the coarseliposomes form smaller similarly closed vesicles.

Water-soluble active ingredients are usually incorporated by dispersingthe cast film with an aqueous solution of the compound. Theunencapsulated compound is then removed by centrifugation,chromatography, dialysis or other art-known suitable procedures. Thelipid-soluble active ingredient is usually incorporated by dissolving itin the organic solvent with the phospholipid prior to casting the film.If the solubility of the material in the lipid phase is not exceeded orthe amount present is not in excess of that which can be bound to thelipid, liposomes prepared by the above method usually contain most ofthe material bound in the lipid bilayers; separation of the liposomesfrom unencapsulated material is not required.

A particularly convenient method for preparing liposome formulated formsof hedgehog and ptc therapeutics is the method described inEP-A-253,619, incorporated herein by reference. In this method, singlebilayered liposomes containing encapsulated active ingredients areprepared by dissolving the lipid component in an organic medium,injecting the organic solution of the lipid component under pressureinto an aqueous component while simultaneously mixing the organic andaqueous components with a high speed homogenizer or mixing means,whereupon the liposomes are formed spontaneously.

The single bilayered liposomes containing the encapsulated hedgehog orptc therapeutic can be employed directly or they can be employed in asuitable pharmaceutically acceptable carrier for topical administration.The viscosity of the liposomes can be increased by the addition of oneor more suitable thickening agents such as, for example xanthan gum,hydroxypropyl cellulose, hydroxypropyl methylcellulose and mixturesthereof. The aqueous component may consist of water alone or it maycontain electrolytes, buffered systems and other ingredients, such as,for example, preservatives. Suitable electrolytes which can be employedinclude metal salts such as alkali metal and alkaline earth metal salts.The preferred metal salts are calcium chloride, sodium chloride andpotassium chloride. The concentration of the electrolyte may vary fromzero to 260 mM, preferably from 5 mM to 160 mM. The aqueous component isplaced in a suitable vessel which can be adapted to effecthomogenization by effecting great turbulence during the injection of theorganic component. Homogenization of the two components can beaccomplished within the vessel, or, alternatively, the aqueous andorganic components may be injected separately into a mixing means whichis located outside the vessel. In the latter case, the liposomes areformed in the mixing means and then transferred to another vessel forcollection purpose.

The organic component consists of a suitable non-toxic, pharmaceuticallyacceptable solvent such as, for example ethanol, glycerol, propyleneglycol and polyethylene glycol, and a suitable phospholipid which issoluble in the solvent. Suitable phospholipids which can be employedinclude lecithin, phosphatidylcholine, phosphatydylserine,phosphatidylethanol-amine, phosphatidylinositol, lysophosphatidylcholineand phospha-tidyl glycerol, for example. Other lipophilic additives maybe employed in order to selectively modify the characteristics of theliposomes. Examples of such other additives include stearylamine,phosphatidic acid, tocopherol, cholesterol and lanolin extracts.

In addition, other ingredients which can prevent oxidation of thephospholipids may be added to the organic component. Examples of suchother ingredients include tocopherol, butylated hydroxyanisole,butylated hydroxytoluene, ascorbyl palmitate and ascorbyl oleate.Preservatives such a benzoic acid, methyl paraben and propyl paraben mayalso be added.

Apart from the above-described compositions, use may be made of covers,e.g. plasters, bandages, dressings, gauze pads and the like, containingan appropriate amount of a hedgehog or ptc therapeutic. In some casesuse may be made of plasters, bandages, dressings, gauze pads and thelike which have been impregnated with a topical formulation containingthe therapeutic formulation.

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Purification of Hedgehog Protein

Human sonic hedgehog protein (residues 24-197) was expressed in thebaculovirus/insect cell system (Roelink et al. (1995) Cell 81:445-455).The conditioned medium was loaded onto Fast Flo SP agarose equilibratedwith 50 mM potassium phosphate, 0.5 mM DTT, pH 7.0. The column waswashed with this buffer, and then eluted with a gradient to 10. M NaCl.Fractions were assayed for the induction of alkaline phosphataseactivity on mesenchymal stem cells (C3H10T1/2 cells, see, e.g., Wang etal. (1993) Growth Factors 9:57-71) and then pooled on the basis of thisactivity and also by purity on SDS gels. The pooled material wasconcentrated on an Amicon ultra filtration unit (PM10 membrane) anddiafiltered against 10 mM Tris, pH 7.4, 0.5 mM DTT. Protein wasestimated by the Bradford method using gamma globulin as a standard.

Preparation of Collagen Sponge

Collagen sponge was washed extensively in MilliQ water to remove anysurfactants and additives from the manufacturer. The sponge was thenwashed in 70% ethanol, then dried in vacuo.

Preparation of Implants

Protein was added to 1.0-1.5 mm by 8-10 mm pieces of collagen sponge(1.5-3.0 mg in weight). In some cases zinc sulfate was added to a finalconcentration of 0.2 mM before the hedgehog protein was added to thecollagen sponge. The reconstituted sponges were then frozen andlyophilized.

Implantation

Sponges were implanted either subcutaneously in the thoracic region ofSprague Dawley rats (4-8 weeks old) or in the thigh muscle of rabbits(11-14 weeks old). Animals were maintained for 2-5 weeks before removingthe implant. The implant was then fixed in 4% formalin or 4%paraformaldehyde and then embedded in JB-4 resin. Sections were stainedwith toluidine blue (Wang et al. (1988) PNAS 85:9484-9488).

The induction of new hair follicles, sebaceous glands, and other dermalstructures were identified by its distinctive morphology. The carefulsubcutaneous or intramuscular placement of our implants and the carefulremoval of these implants preclude the possibility of contamination fromexisting dermal structures. Also, the appearance of more immature hairfollicles is seen in the implants of shorter (2 week) duration.

Biopsy slides were obtained from an intramuscular implant taken out of arabbit muscle at three weeks and stained with hematoxylin and eosin.Similar slides were examined of an intramuscular implant, rabbit muscle,three weeks, stained with toluidine blue. Slides of certain samplesrevealed a tissue morphology indicating the presence of follicle- andhair-like structures forming in the intramuscular tissue.

Hair Induction by Shh

As a follow-up to the above experiments, hedgehog-loaded collagensponges were implanted under the shaved skin of mice. As indicated inFIGS. 1A-C, the hedgehog preparations were able to induce hair growthover the implants. Moreover, Ihh protein modified at the C terminus witha Von Willebrand's factor collagen binding site was active in hairgrowth, indicating a localized inducing activity of the implantedprotein.

All of the above-cited references and publications are herebyincorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific polypeptides, nucleic acids, methods, assays and reagentsdescribed herein. Such equivalents are considered to be within the scopeof this invention.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 28 <210> SEQ ID NO 1 <211> LENGTH: 1277<212> TYPE: DNA <213> ORGANISM: chicken Shh <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(1275) <400> SEQUENCE: 1atg gtc gaa atg ctg ctg ttg aca aga att ct#c ttg gtg ggc ttc atc       48Met Val Glu Met Leu Leu Leu Thr Arg Ile Le #u Leu Val Gly Phe Ile  1               5  #                 10  #                 15tgc gct ctt tta gtc tcc tct ggg ctg act tg#t gga cca ggc agg ggc       96Cys Ala Leu Leu Val Ser Ser Gly Leu Thr Cy #s Gly Pro Gly Arg Gly             20      #             25      #             30att gga aaa agg agg cac ccc aaa aag ctg ac#c ccg tta gcc tat aag      144Ile Gly Lys Arg Arg His Pro Lys Lys Leu Th #r Pro Leu Ala Tyr Lys         35          #         40          #         45cag ttt att ccc aat gtg gca gag aag acc ct#a ggg gcc agt gga aga      192Gln Phe Ile Pro Asn Val Ala Glu Lys Thr Le #u Gly Ala Ser Gly Arg     50              #     55              #     60tat gaa ggg aag atc aca aga aac tcc gag ag#a ttt aaa gaa cta acc      240Tyr Glu Gly Lys Ile Thr Arg Asn Ser Glu Ar #g Phe Lys Glu Leu Thr 65                  # 70                  # 75                  # 80cca aat tac aac cct gac att att ttt aag ga#t gaa gag aac acg gga      288Pro Asn Tyr Asn Pro Asp Ile Ile Phe Lys As #p Glu Glu Asn Thr Gly                 85  #                 90  #                 95gct gac aga ctg atg act cag cgc tgc aag ga#c aag ctg aat gcc ctg      336Ala Asp Arg Leu Met Thr Gln Arg Cys Lys As #p Lys Leu Asn Ala Leu            100       #           105       #           110gcg atc tcg gtg atg aac cag tgg ccc ggg gt#g aag ctg cgg gtg acc      384Ala Ile Ser Val Met Asn Gln Trp Pro Gly Va #l Lys Leu Arg Val Thr        115           #       120           #       125gag ggc tgg gac gag gat ggc cat cac tcc ga#g gaa tcg ctg cac tac      432Glu Gly Trp Asp Glu Asp Gly His His Ser Gl #u Glu Ser Leu His Tyr    130               #   135               #   140gag ggt cgc gcc gtg gac atc acc acg tcg ga#t cgg gac cgc agc aag      480Glu Gly Arg Ala Val Asp Ile Thr Thr Ser As #p Arg Asp Arg Ser Lys145                 1 #50                 1 #55                 1 #60tac gga atg ctg gcc cgc ctc gcc gtc gag gc#c ggc ttc gac tgg gtc      528Tyr Gly Met Leu Ala Arg Leu Ala Val Glu Al #a Gly Phe Asp Trp Val                165   #               170   #               175tac tac gag tcc aag gcg cac atc cac tgc tc#c gtc aaa gca gaa aac      576Tyr Tyr Glu Ser Lys Ala His Ile His Cys Se #r Val Lys Ala Glu Asn            180       #           185       #           190tca gtg gca gcg aaa tca gga ggc tgc ttc cc#t ggc tca gcc aca gtg      624Ser Val Ala Ala Lys Ser Gly Gly Cys Phe Pr #o Gly Ser Ala Thr Val        195           #       200           #       205cac ctg gag cat gga ggc acc aag ctg gtg aa#g gac ctg agc cct ggg      672His Leu Glu His Gly Gly Thr Lys Leu Val Ly #s Asp Leu Ser Pro Gly    210               #   215               #   220gac cgc gtg ctg gct gct gac gcg gac ggc cg#g ctg ctc tac agt gac      720Asp Arg Val Leu Ala Ala Asp Ala Asp Gly Ar #g Leu Leu Tyr Ser Asp225                 2 #30                 2 #35                 2 #40ttc ctc acc ttc ctc gac cgg atg gac agc tc#c cga aag ctc ttc tac      768Phe Leu Thr Phe Leu Asp Arg Met Asp Ser Se #r Arg Lys Leu Phe Tyr                245   #               250   #               255gtc atc gag acg cgg cag ccc cgg gcc cgg ct#g cta ctg acg gcg gcc      816Val Ile Glu Thr Arg Gln Pro Arg Ala Arg Le #u Leu Leu Thr Ala Ala            260       #           265       #           270cac ctg ctc ttt gtg gcc ccc cag cac aac ca#g tcg gag gcc aca ggg      864His Leu Leu Phe Val Ala Pro Gln His Asn Gl #n Ser Glu Ala Thr Gly        275           #       280           #       285tcc acc agt ggc cag gcg ctc ttc gcc agc aa#c gtg aag cct ggc caa      912Ser Thr Ser Gly Gln Ala Leu Phe Ala Ser As #n Val Lys Pro Gly Gln    290               #   295               #   300cgt gtc tat gtg ctg ggc gag ggc ggg cag ca#g ctg ctg ccg gcg tct      960Arg Val Tyr Val Leu Gly Glu Gly Gly Gln Gl #n Leu Leu Pro Ala Ser305                 3 #10                 3 #15                 3 #20gtc cac agc gtc tca ttg cgg gag gag gcg tc#c gga gcc tac gcc cca     1008Val His Ser Val Ser Leu Arg Glu Glu Ala Se #r Gly Ala Tyr Ala Pro                325   #               330   #               335ctc acc gcc cag ggc acc atc ctc atc aac cg#g gtg ttg gcc tcc tgc     1056Leu Thr Ala Gln Gly Thr Ile Leu Ile Asn Ar #g Val Leu Ala Ser Cys            340       #           345       #           350tac gcc gtc atc gag gag cac agt tgg gcc ca#t tgg gcc ttc gca cca     1104Tyr Ala Val Ile Glu Glu His Ser Trp Ala Hi #s Trp Ala Phe Ala Pro        355           #       360           #       365ttc cgc ttg gct cag ggg ctg ctg gcc gcc ct#c tgc cca gat ggg gcc     1152Phe Arg Leu Ala Gln Gly Leu Leu Ala Ala Le #u Cys Pro Asp Gly Ala    370               #   375               #   380atc cct act gcc gcc acc acc acc act ggc at#c cat tgg tac tca cgg     1200Ile Pro Thr Ala Ala Thr Thr Thr Thr Gly Il #e His Trp Tyr Ser Arg385                 3 #90                 3 #95                 4 #00ctc ctc tac cgc atc ggc agc tgg gtg ctg ga#t ggt gac gcg ctg cat     1248Leu Leu Tyr Arg Ile Gly Ser Trp Val Leu As #p Gly Asp Ala Leu His                405   #               410   #               415ccg ctg ggc atg gtg gca ccg gcc agc tg   #                  #          1277 Pro Leu Gly Met Val Ala Pro Ala Ser            420       #           425 <210> SEQ ID NO 2<211> LENGTH: 1190 <212> TYPE: DNA <213> ORGANISM: murine Dhh<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1188)<400> SEQUENCE: 2 atg gct ctg ccg gcc agt ctg ttg ccc ctg tg#c tgc ttg gca ctc ttg       48Met Ala Leu Pro Ala Ser Leu Leu Pro Leu Cy #s Cys Leu Ala Leu Leu  1               5  #                 10  #                 15gca cta tct gcc cag agc tgc ggg ccg ggc cg#a gga ccg gtt ggc cgg       96Ala Leu Ser Ala Gln Ser Cys Gly Pro Gly Ar #g Gly Pro Val Gly Arg             20      #             25      #             30cgg cgt tat gtg cgc aag caa ctt gtg cct ct#g cta tac aag cag ttt      144Arg Arg Tyr Val Arg Lys Gln Leu Val Pro Le #u Leu Tyr Lys Gln Phe         35          #         40          #         45gtg ccc agt atg ccc gag cgg acc ctg ggc gc#g agt ggg cca gcg gag      192Val Pro Ser Met Pro Glu Arg Thr Leu Gly Al #a Ser Gly Pro Ala Glu     50              #     55              #     60ggg agg gta aca agg ggg tcg gag cgc ttc cg#g gac ctc gta ccc aac      240Gly Arg Val Thr Arg Gly Ser Glu Arg Phe Ar #g Asp Leu Val Pro Asn 65                  # 70                  # 75                  # 80tac aac ccc gac ata atc ttc aag gat gag ga#g aac agc ggc gca gac      288Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Gl #u Asn Ser Gly Ala Asp                 85  #                 90  #                 95cgc ctg atg aca gag cgt tgc aaa gag cgg gt#g aac gct cta gcc atc      336Arg Leu Met Thr Glu Arg Cys Lys Glu Arg Va #l Asn Ala Leu Ala Ile            100       #           105       #           110gcg gtg atg aac atg tgg ccc gga gta cgc ct#a cgt gtg act gaa ggc      384Ala Val Met Asn Met Trp Pro Gly Val Arg Le #u Arg Val Thr Glu Gly        115           #       120           #       125tgg gac gag gac ggc cac cac gca cag gat tc#a ctc cac tac gaa ggc      432Trp Asp Glu Asp Gly His His Ala Gln Asp Se #r Leu His Tyr Glu Gly    130               #   135               #   140cgt gcc ttg gac atc acc acg tct gac cgt ga#c cgt aat aag tat ggt      480Arg Ala Leu Asp Ile Thr Thr Ser Asp Arg As #p Arg Asn Lys Tyr Gly145                 1 #50                 1 #55                 1 #60ttg ttg gcg cgc cta gct gtg gaa gcc gga tt#c gac tgg gtc tac tac      528Leu Leu Ala Arg Leu Ala Val Glu Ala Gly Ph #e Asp Trp Val Tyr Tyr                165   #               170   #               175gag tcc cgc aac cac atc cac gta tcg gtc aa#a gct gat aac tca ctg      576Glu Ser Arg Asn His Ile His Val Ser Val Ly #s Ala Asp Asn Ser Leu            180       #           185       #           190gcg gtc cga gcc gga ggc tgc ttt ccg gga aa#t gcc acg gtg cgc ttg      624Ala Val Arg Ala Gly Gly Cys Phe Pro Gly As #n Ala Thr Val Arg Leu        195           #       200           #       205cgg agc ggc gaa cgg aag ggg ctg agg gaa ct#a cat cgt ggt gac tgg      672Arg Ser Gly Glu Arg Lys Gly Leu Arg Glu Le #u His Arg Gly Asp Trp    210               #   215               #   220gta ctg gcc gct gat gca gcg ggc cga gtg gt#a ccc acg cca gtg ctg      720Val Leu Ala Ala Asp Ala Ala Gly Arg Val Va #l Pro Thr Pro Val Leu225                 2 #30                 2 #35                 2 #40ctc ttc ctg gac cgg gat ctg cag cgc cgc gc#c tcg ttc gtg gct gtg      768Leu Phe Leu Asp Arg Asp Leu Gln Arg Arg Al #a Ser Phe Val Ala Val                245   #               250   #               255gag acc gag cgg cct ccg cgc aaa ctg ttg ct#c aca ccc tgg cat ctg      816Glu Thr Glu Arg Pro Pro Arg Lys Leu Leu Le #u Thr Pro Trp His Leu            260       #           265       #           270gtg ttc gct gct cgc ggg cca gcg cct gct cc#a ggt gac ttt gca ccg      864Val Phe Ala Ala Arg Gly Pro Ala Pro Ala Pr #o Gly Asp Phe Ala Pro        275           #       280           #       285gtg ttc gcg cgc cgc tta cgt gct ggc gac tc#g gtg ctg gct ccc ggc      912Val Phe Ala Arg Arg Leu Arg Ala Gly Asp Se #r Val Leu Ala Pro Gly    290               #   295               #   300ggg gac gcg ctc cag ccg gcg cgc gta gcc cg#c gtg gcg cgc gag gaa      960Gly Asp Ala Leu Gln Pro Ala Arg Val Ala Ar #g Val Ala Arg Glu Glu305                 3 #10                 3 #15                 3 #20gcc gtg ggc gtg ttc gca ccg ctc act gcg ca#c ggg acg ctg ctg gtc     1008Ala Val Gly Val Phe Ala Pro Leu Thr Ala Hi #s Gly Thr Leu Leu Val                325   #               330   #               335aac gac gtc ctc gcc tcc tgc tac gcg gtt ct#a gag agt cac cag tgg     1056Asn Asp Val Leu Ala Ser Cys Tyr Ala Val Le #u Glu Ser His Gln Trp            340       #           345       #           350gcc cac cgc gcc ttc gcc cct ttg cgg ctg ct#g cac gcg ctc ggg gct     1104Ala His Arg Ala Phe Ala Pro Leu Arg Leu Le #u His Ala Leu Gly Ala        355           #       360           #       365ctg ctc cct ggg ggt gca gtc cag ccg act gg#c atg cat tgg tac tct     1152Leu Leu Pro Gly Gly Ala Val Gln Pro Thr Gl #y Met His Trp Tyr Ser    370               #   375               #   380cgc ctc ctt tac cgc ttg gcc gag gag tta at #g ggc tg               #   1190 Arg Leu Leu Tyr Arg Leu Ala Glu Glu Leu Me #t Gly385                 3 #90                 3 #95 <210> SEQ ID NO 3<211> LENGTH: 1281 <212> TYPE: DNA <213> ORGANISM: murine Ihh<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1233)<400> SEQUENCE: 3 atg tct ccc gcc tgg ctc cgg ccc cga ctg cg#g ttc tgt ctg ttc ctg       48Met Ser Pro Ala Trp Leu Arg Pro Arg Leu Ar #g Phe Cys Leu Phe Leu  1               5  #                 10  #                 15ctg ctg ctg ctt ctg gtg ccg gcg gcg cgg gg#c tgc ggg ccg ggc cgg       96Leu Leu Leu Leu Leu Val Pro Ala Ala Arg Gl #y Cys Gly Pro Gly Arg             20      #             25      #             30gtg gtg ggc agc cgc cgg agg ccg cct cgc aa#g ctc gtg cct ctt gcc      144Val Val Gly Ser Arg Arg Arg Pro Pro Arg Ly #s Leu Val Pro Leu Ala         35          #         40          #         45tac aag cag ttc agc ccc aac gtg ccg gag aa#g acc ctg ggc gcc agc      192Tyr Lys Gln Phe Ser Pro Asn Val Pro Glu Ly #s Thr Leu Gly Ala Ser     50              #     55              #     60ggg cgc tac gaa ggc aag atc gcg cgc agc tc#t gag cgc ttc aaa gag      240Gly Arg Tyr Glu Gly Lys Ile Ala Arg Ser Se #r Glu Arg Phe Lys Glu 65                  # 70                  # 75                  # 80ctc acc ccc aac tac aat ccc gac atc atc tt#c aag gac gag gag aac      288Leu Thr Pro Asn Tyr Asn Pro Asp Ile Ile Ph #e Lys Asp Glu Glu Asn                 85  #                 90  #                 95acg ggt gcc gac cgc ctc atg acc cag cgc tg#c aag gac cgt ctg aac      336Thr Gly Ala Asp Arg Leu Met Thr Gln Arg Cy #s Lys Asp Arg Leu Asn            100       #           105       #           110tca ctg gcc atc tct gtc atg aac cag tgg cc#t ggt gtg aaa ctg cgg      384Ser Leu Ala Ile Ser Val Met Asn Gln Trp Pr #o Gly Val Lys Leu Arg        115           #       120           #       125gtg acc gaa ggc cgg gat gaa gat ggc cat ca#c tca gag gag tct tta      432Val Thr Glu Gly Arg Asp Glu Asp Gly His Hi #s Ser Glu Glu Ser Leu    130               #   135               #   140cac tat gag ggc cgc gcg gtg gat atc acc ac#c tca gac cgt gac cga      480His Tyr Glu Gly Arg Ala Val Asp Ile Thr Th #r Ser Asp Arg Asp Arg145                 1 #50                 1 #55                 1 #60aat aag tat gga ctg ctg gcg cgc tta gca gt#g gag gcc ggc ttc gac      528Asn Lys Tyr Gly Leu Leu Ala Arg Leu Ala Va #l Glu Ala Gly Phe Asp                165   #               170   #               175tgg gtg tat tac gag tcc aag gcc cac gtg ca#t tgc tct gtc aag tct      576Trp Val Tyr Tyr Glu Ser Lys Ala His Val Hi #s Cys Ser Val Lys Ser            180       #           185       #           190gag cat tcg gcc gct gcc aag aca ggt ggc tg#c ttt cct gcc gga gcc      624Glu His Ser Ala Ala Ala Lys Thr Gly Gly Cy #s Phe Pro Ala Gly Ala        195           #       200           #       205cag gtg cgc cta gag aac ggg gag cgt gtg gc#c ctg tca gct gta aag      672Gln Val Arg Leu Glu Asn Gly Glu Arg Val Al #a Leu Ser Ala Val Lys    210               #   215               #   220cca gga gac cgg gtg ctg gcc atg ggg gag ga#t ggg acc ccc acc ttc      720Pro Gly Asp Arg Val Leu Ala Met Gly Glu As #p Gly Thr Pro Thr Phe225                 2 #30                 2 #35                 2 #40agt gat gtg ctt att ttc ctg gac cgc gag cc#a aac cgg ctg aga gct      768Ser Asp Val Leu Ile Phe Leu Asp Arg Glu Pr #o Asn Arg Leu Arg Ala                245   #               250   #               255ttc cag gtc atc gag act cag gat cct ccg cg#t cgg ctg gcg ctc acg      816Phe Gln Val Ile Glu Thr Gln Asp Pro Pro Ar #g Arg Leu Ala Leu Thr            260       #           265       #           270cct gcc cac ctg ctc ttc att gcg gac aat ca#t aca gaa cca gca gcc      864Pro Ala His Leu Leu Phe Ile Ala Asp Asn Hi #s Thr Glu Pro Ala Ala        275           #       280           #       285cac ttc cgg gcc aca ttt gcc agc cat gtg ca#a cca ggc caa tat gtg      912His Phe Arg Ala Thr Phe Ala Ser His Val Gl #n Pro Gly Gln Tyr Val    290               #   295               #   300ctg gta tca ggg gta cca ggc ctc cag cct gc#t cgg gtg gca gct gtc      960Leu Val Ser Gly Val Pro Gly Leu Gln Pro Al #a Arg Val Ala Ala Val305                 3 #10                 3 #15                 3 #20tcc acc cac gtg gcc ctt ggg tcc tat gct cc#t ctc aca agg cat ggg     1008Ser Thr His Val Ala Leu Gly Ser Tyr Ala Pr #o Leu Thr Arg His Gly                325   #               330   #               335aca ctt gtg gtg gag gat gtg gtg gcc tcc tg#c ttt gca gct gtg gct     1056Thr Leu Val Val Glu Asp Val Val Ala Ser Cy #s Phe Ala Ala Val Ala            340       #           345       #           350gac cac cat ctg gct cag ttg gcc ttc tgg cc#c ctg cga ctg ttt ccc     1104Asp His His Leu Ala Gln Leu Ala Phe Trp Pr #o Leu Arg Leu Phe Pro        355           #       360           #       365agt ttg gca tgg ggc agc tgg acc cca agt ga#g ggt gtt cac tcc tac     1152Ser Leu Ala Trp Gly Ser Trp Thr Pro Ser Gl #u Gly Val His Ser Tyr    370               #   375               #   380cct cag atg ctc tac cgc ctg ggg cgt ctc tt#g cta gaa gag agc acc     1200Pro Gln Met Leu Tyr Arg Leu Gly Arg Leu Le #u Leu Glu Glu Ser Thr385                 3 #90                 3 #95                 4 #00ttc cat cca ctg ggc atg tct ggg gca gga ag#c tgaagggact ctaaccactg   1253Phe His Pro Leu Gly Met Ser Gly Ala Gly Se #r                 405  #               410 ccctcctgga actgctgtgc gtggatcc         #                   #           1281 <210> SEQ ID NO 4<211> LENGTH: 1313 <212> TYPE: DNA <213> ORGANISM: murine Shh<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1311)<400> SEQUENCE: 4 atg ctg ctg ctg ctg gcc aga tgt ttt ctg gt#g atc ctt gct tcc tcg       48Met Leu Leu Leu Leu Ala Arg Cys Phe Leu Va #l Ile Leu Ala Ser Ser  1               5  #                 10  #                 15ctg ctg gtg tgc ccc ggg ctg gcc tgt ggg cc#c ggc agg ggg ttt gga       96Leu Leu Val Cys Pro Gly Leu Ala Cys Gly Pr #o Gly Arg Gly Phe Gly             20      #             25      #             30aag agg cgg cac ccc aaa aag ctg acc cct tt#a gcc tac aag cag ttt      144Lys Arg Arg His Pro Lys Lys Leu Thr Pro Le #u Ala Tyr Lys Gln Phe         35          #         40          #         45att ccc aac gta gcc gag aag acc cta ggg gc#c agc ggc aga tat gaa      192Ile Pro Asn Val Ala Glu Lys Thr Leu Gly Al #a Ser Gly Arg Tyr Glu     50              #     55              #     60ggg aag atc aca aga aac tcc gaa cga ttt aa#g gaa ctc acc ccc aat      240Gly Lys Ile Thr Arg Asn Ser Glu Arg Phe Ly #s Glu Leu Thr Pro Asn 65                  # 70                  # 75                  # 80tac aac ccc gac atc ata ttt aag gat gag ga#a aac acg gga gca gac      288Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Gl #u Asn Thr Gly Ala Asp                 85  #                 90  #                 95cgg ctg atg act cag agg tgc aaa gac aag tt#a aat gcc ttg gcc atc      336Arg Leu Met Thr Gln Arg Cys Lys Asp Lys Le #u Asn Ala Leu Ala Ile            100       #           105       #           110tct gtg atg aac cag tgg cct gga gtg agg ct#g cga gtg acc gag ggc      384Ser Val Met Asn Gln Trp Pro Gly Val Arg Le #u Arg Val Thr Glu Gly        115           #       120           #       125tgg gat gag gac ggc cat cat tca gag gag tc#t cta cac tat gag ggt      432Trp Asp Glu Asp Gly His His Ser Glu Glu Se #r Leu His Tyr Glu Gly    130               #   135               #   140cga gca gtg gac atc acc acg tcc gac cgg ga#c cgc agc aag tac ggc      480Arg Ala Val Asp Ile Thr Thr Ser Asp Arg As #p Arg Ser Lys Tyr Gly145                 1 #50                 1 #55                 1 #60atg ctg gct cgc ctg gct gtg gaa gca ggt tt#c gac tgg gtc tac tat      528Met Leu Ala Arg Leu Ala Val Glu Ala Gly Ph #e Asp Trp Val Tyr Tyr                165   #               170   #               175gaa tcc aaa gct cac atc cac tgt tct gtg aa#a gca gag aac tcc gtg      576Glu Ser Lys Ala His Ile His Cys Ser Val Ly #s Ala Glu Asn Ser Val            180       #           185       #           190gcg gcc aaa tcc ggc ggc tgt ttc ccg gga tc#c gcc acc gtg cac ctg      624Ala Ala Lys Ser Gly Gly Cys Phe Pro Gly Se #r Ala Thr Val His Leu        195           #       200           #       205gag cag ggc ggc acc aag ctg gtg aag gac tt#a cgt ccc gga gac cgc      672Glu Gln Gly Gly Thr Lys Leu Val Lys Asp Le #u Arg Pro Gly Asp Arg    210               #   215               #   220gtg ctg gcg gct gac gac cag ggc cgg ctg ct#g tac agc gac ttc ctc      720Val Leu Ala Ala Asp Asp Gln Gly Arg Leu Le #u Tyr Ser Asp Phe Leu225                 2 #30                 2 #35                 2 #40acc ttc ctg gac cgc gac gaa ggc gcc aag aa#g gtc ttc tac gtg atc      768Thr Phe Leu Asp Arg Asp Glu Gly Ala Lys Ly #s Val Phe Tyr Val Ile                245   #               250   #               255gag acg ctg gag ccg cgc gag cgc ctg ctg ct#c acc gcc gcg cac ctg      816Glu Thr Leu Glu Pro Arg Glu Arg Leu Leu Le #u Thr Ala Ala His Leu            260       #           265       #           270ctc ttc gtg gcg ccg cac aac gac tcg ggg cc#c acg ccc ggg cca agc      864Leu Phe Val Ala Pro His Asn Asp Ser Gly Pr #o Thr Pro Gly Pro Ser        275           #       280           #       285gcg ctc ttt gcc agc cgc gtg cgc ccc ggg ca#g cgc gtg tac gtg gtg      912Ala Leu Phe Ala Ser Arg Val Arg Pro Gly Gl #n Arg Val Tyr Val Val    290               #   295               #   300gct gaa cgc ggc ggg gac cgc cgg ctg ctg cc#c gcc gcg gtg cac agc      960Ala Glu Arg Gly Gly Asp Arg Arg Leu Leu Pr #o Ala Ala Val His Ser305                 3 #10                 3 #15                 3 #20gtg acg ctg cga gag gag gag gcg ggc gcg ta#c gcg ccg ctc acg gcg     1008Val Thr Leu Arg Glu Glu Glu Ala Gly Ala Ty #r Ala Pro Leu Thr Ala                325   #               330   #               335cac ggc acc att ctc atc aac cgg gtg ctc gc#c tcg tgc tac gct gtc     1056His Gly Thr Ile Leu Ile Asn Arg Val Leu Al #a Ser Cys Tyr Ala Val            340       #           345       #           350atc gag gag cac agc tgg gca cac cgg gcc tt#c gcg cct ttc cgc ctg     1104Ile Glu Glu His Ser Trp Ala His Arg Ala Ph #e Ala Pro Phe Arg Leu        355           #       360           #       365gcg cac gcg ctg ctg gcc gcg ctg gca ccc gc#c cgc acg gac ggc ggg     1152Ala His Ala Leu Leu Ala Ala Leu Ala Pro Al #a Arg Thr Asp Gly Gly    370               #   375               #   380ggc ggg ggc agc atc cct gca gcg caa tct gc#a acg gaa gcg agg ggc     1200Gly Gly Gly Ser Ile Pro Ala Ala Gln Ser Al #a Thr Glu Ala Arg Gly385                 3 #90                 3 #95                 4 #00gcg gag ccg act gcg ggc atc cac tgg tac tc#g cag ctg ctc tac cac     1248Ala Glu Pro Thr Ala Gly Ile His Trp Tyr Se #r Gln Leu Leu Tyr His                405   #               410   #               415att ggc acc tgg ctg ttg gac agc gag acc at#g cat ccc ttg gga atg     1296Ile Gly Thr Trp Leu Leu Asp Ser Glu Thr Me #t His Pro Leu Gly Met            420       #           425       #           430gcg gtc aag tcc agc tg          #                   #                  # 1313 Ala Val Lys Ser Ser         435 <210> SEQ ID NO 5<211> LENGTH: 1256 <212> TYPE: DNA <213> ORGANISM: zebrafish Shh<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1254)<400> SEQUENCE: 5 atg cgg ctt ttg acg aga gtg ctg ctg gtg tc#t ctt ctc act ctg tcc       48Met Arg Leu Leu Thr Arg Val Leu Leu Val Se #r Leu Leu Thr Leu Ser  1               5  #                 10  #                 15ttg gtg gtg tcc gga ctg gcc tgc ggt cct gg#c aga ggc tac ggc aga       96Leu Val Val Ser Gly Leu Ala Cys Gly Pro Gl #y Arg Gly Tyr Gly Arg             20      #             25      #             30aga aga cat ccg aag aag ctg aca cct ctc gc#c tac aag cag ttc ata      144Arg Arg His Pro Lys Lys Leu Thr Pro Leu Al #a Tyr Lys Gln Phe Ile         35          #         40          #         45cct aat gtc gcg gag aag acc tta ggg gcc ag#c ggc aga tac gag ggc      192Pro Asn Val Ala Glu Lys Thr Leu Gly Ala Se #r Gly Arg Tyr Glu Gly     50              #     55              #     60aag ata acg cgc aat tcg gag aga ttt aaa ga#a ctt act cca aat tac      240Lys Ile Thr Arg Asn Ser Glu Arg Phe Lys Gl #u Leu Thr Pro Asn Tyr 65                  # 70                  # 75                  # 80aat ccc gac att atc ttt aag gat gag gag aa#c acg gga gcg gac agg      288Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu As #n Thr Gly Ala Asp Arg                 85  #                 90  #                 95ctc atg aca cag aga tgc aaa gac aag ctg aa#c tcg ctg gcc atc tct      336Leu Met Thr Gln Arg Cys Lys Asp Lys Leu As #n Ser Leu Ala Ile Ser            100       #           105       #           110gta atg aac cac tgg cca ggg gtt aag ctg cg#t gtg aca gag ggc tgg      384Val Met Asn His Trp Pro Gly Val Lys Leu Ar #g Val Thr Glu Gly Trp        115           #       120           #       125gat gag gac ggt cac cat ttt gaa gaa tca ct#c cac tac gag gga aga      432Asp Glu Asp Gly His His Phe Glu Glu Ser Le #u His Tyr Glu Gly Arg    130               #   135               #   140gct gtt gat att acc acc tct gac cga gac aa#g agc aaa tac ggg aca      480Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Ly #s Ser Lys Tyr Gly Thr145                 1 #50                 1 #55                 1 #60ctg tct cgc cta gct gtg gag gct gga ttt ga#c tgg gtc tat tac gag      528Leu Ser Arg Leu Ala Val Glu Ala Gly Phe As #p Trp Val Tyr Tyr Glu                165   #               170   #               175tcc aaa gcc cac att cat tgc tct gtc aaa gc#a gaa aat tcg gtt gct      576Ser Lys Ala His Ile His Cys Ser Val Lys Al #a Glu Asn Ser Val Ala            180       #           185       #           190gcg aaa tct ggg ggc tgt ttc cca ggt tcg gc#t ctg gtc tcg ctc cag      624Ala Lys Ser Gly Gly Cys Phe Pro Gly Ser Al #a Leu Val Ser Leu Gln        195           #       200           #       205gac gga gga cag aag gcc gtg aag gac ctg aa#c ccc gga gac aag gtg      672Asp Gly Gly Gln Lys Ala Val Lys Asp Leu As #n Pro Gly Asp Lys Val    210               #   215               #   220ctg gcg gca gac agc gcg gga aac ctg gtg tt#c agc gac ttc atc atg      720Leu Ala Ala Asp Ser Ala Gly Asn Leu Val Ph #e Ser Asp Phe Ile Met225                 2 #30                 2 #35                 2 #40ttc aca gac cga gac tcc acg acg cga cgt gt#g ttt tac gtc ata gaa      768Phe Thr Asp Arg Asp Ser Thr Thr Arg Arg Va #l Phe Tyr Val Ile Glu                245   #               250   #               255acg caa gaa ccc gtt gaa aag atc acc ctc ac#c gcc gct cac ctc ctt      816Thr Gln Glu Pro Val Glu Lys Ile Thr Leu Th #r Ala Ala His Leu Leu            260       #           265       #           270ttt gtc ctc gac aac tca acg gaa gat ctc ca#c acc atg acc gcc gcg      864Phe Val Leu Asp Asn Ser Thr Glu Asp Leu Hi #s Thr Met Thr Ala Ala        275           #       280           #       285tat gcc agc agt gtc aga gcc gga caa aag gt#g atg gtt gtt gat gat      912Tyr Ala Ser Ser Val Arg Ala Gly Gln Lys Va #l Met Val Val Asp Asp    290               #   295               #   300agc ggt cag ctt aaa tct gtc atc gtg cag cg#g ata tac acg gag gag      960Ser Gly Gln Leu Lys Ser Val Ile Val Gln Ar #g Ile Tyr Thr Glu Glu305                 3 #10                 3 #15                 3 #20cag cgg ggc tcg ttc gca cca gtg act gca ca#t ggg acc att gtg gtc     1008Gln Arg Gly Ser Phe Ala Pro Val Thr Ala Hi #s Gly Thr Ile Val Val                325   #               330   #               335gac aga ata ctg gcg tcc tgt tac gcc gta at#a gag gac cag ggg ctt     1056Asp Arg Ile Leu Ala Ser Cys Tyr Ala Val Il #e Glu Asp Gln Gly Leu            340       #           345       #           350gcg cat ttg gcc ttc gcg ccc gcc agg ctc ta#t tat tac gtg tca tca     1104Ala His Leu Ala Phe Ala Pro Ala Arg Leu Ty #r Tyr Tyr Val Ser Ser        355           #       360           #       365ttc ctg tcc ccc aaa act cca gca gtc ggt cc#a atg cga ctt tac aac     1152Phe Leu Ser Pro Lys Thr Pro Ala Val Gly Pr #o Met Arg Leu Tyr Asn    370               #   375               #   380agg agg ggg tcc act ggt act cca ggc tcc tg#t cat caa atg gga acg     1200Arg Arg Gly Ser Thr Gly Thr Pro Gly Ser Cy #s His Gln Met Gly Thr385                 3 #90                 3 #95                 4 #00tgg ctt ttg gac agc aac atg ctt cat cct tt#g ggg atg tca gta aac     1248Trp Leu Leu Asp Ser Asn Met Leu His Pro Le #u Gly Met Ser Val Asn                405   #               410   #               415tca agc tg               #                   #                  #        1256 Ser Ser <210> SEQ ID NO 6 <211> LENGTH: 1425<212> TYPE: DNA <213> ORGANISM: Homo sapien Shh <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(1425) <220> FEATURE:<223> OTHER INFORMATION: “nnn” encoding “Xaa#” at position 1387-1389 may       be a, t, c, g, other or unk #nown<400> SEQUENCE: 6 atg ctg ctg ctg gcg aga tgt ctg ctg cta gt#c ctc gtc tcc tcg ctg       48Met Leu Leu Leu Ala Arg Cys Leu Leu Leu Va #l Leu Val Ser Ser Leu  1               5  #                 10  #                 15ctg gta tgc tcg gga ctg gcg tgc gga ccg gg#c agg ggg ttc ggg aag       96Leu Val Cys Ser Gly Leu Ala Cys Gly Pro Gl #y Arg Gly Phe Gly Lys             20      #             25      #             30agg agg cac ccc aaa aag ctg acc cct tta gc#c tac aag cag ttt atc      144Arg Arg His Pro Lys Lys Leu Thr Pro Leu Al #a Tyr Lys Gln Phe Ile         35          #         40          #         45ccc aat gtg gcc gag aag acc cta ggc gcc ag#c gga agg tat gaa ggg      192Pro Asn Val Ala Glu Lys Thr Leu Gly Ala Se #r Gly Arg Tyr Glu Gly     50              #     55              #     60aag atc tcc aga aac tcc gag cga ttt aag ga#a ctc acc ccc aat tac      240Lys Ile Ser Arg Asn Ser Glu Arg Phe Lys Gl #u Leu Thr Pro Asn Tyr 65                  # 70                  # 75                  # 80aac ccc gac atc ata ttt aag gat gaa gaa aa#c acc gga gcg gac agg      288Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu As #n Thr Gly Ala Asp Arg                 85  #                 90  #                 95ctg atg act cag agg tgt aag gac aag ttg aa#c gct ttg gcc atc tcg      336Leu Met Thr Gln Arg Cys Lys Asp Lys Leu As #n Ala Leu Ala Ile Ser            100       #           105       #           110gtg atg aac cag tgg cca gga gtg aaa ctg cg#g gtg acc gag ggc tgg      384Val Met Asn Gln Trp Pro Gly Val Lys Leu Ar #g Val Thr Glu Gly Trp        115           #       120           #       125gac gaa gat ggc cac cac tca gag gag tct ct#g cac tac gag ggc cgc      432Asp Glu Asp Gly His His Ser Glu Glu Ser Le #u His Tyr Glu Gly Arg    130               #   135               #   140gca gtg gac atc acc acg tct gac cgc gac cg#c agc aag tac ggc atg      480Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Ar #g Ser Lys Tyr Gly Met145                 1 #50                 1 #55                 1 #60ctg gcc cgc ctg gcg gtg gag gcc ggc ttc ga#c tgg gtg tac tac gag      528Leu Ala Arg Leu Ala Val Glu Ala Gly Phe As #p Trp Val Tyr Tyr Glu                165   #               170   #               175tcc aag gca cat atc cac tgc tcg gtg aaa gc#a gag aac tcg gtg gcg      576Ser Lys Ala His Ile His Cys Ser Val Lys Al #a Glu Asn Ser Val Ala            180       #           185       #           190gcc aaa tcg gga ggc tgc ttc ccg ggc tcg gc#c acg gtg cac ctg gag      624Ala Lys Ser Gly Gly Cys Phe Pro Gly Ser Al #a Thr Val His Leu Glu        195           #       200           #       205cag ggc ggc acc aag ctg gtg aag gac ctg ag#c ccc ggg gac cgc gtg      672Gln Gly Gly Thr Lys Leu Val Lys Asp Leu Se #r Pro Gly Asp Arg Val    210               #   215               #   220ctg gcg gcg gac gac cag ggc cgg ctg ctc ta#c agc gac ttc ctc act      720Leu Ala Ala Asp Asp Gln Gly Arg Leu Leu Ty #r Ser Asp Phe Leu Thr225                 2 #30                 2 #35                 2 #40ttc ctg gac cgc gac gac ggc gcc aag aag gt#c ttc tac gtg atc gag      768Phe Leu Asp Arg Asp Asp Gly Ala Lys Lys Va #l Phe Tyr Val Ile Glu                245   #               250   #               255acg cgg gag ccg cgc gag cgc ctg ctg ctc ac#c gcc gcg cac ctg ctc      816Thr Arg Glu Pro Arg Glu Arg Leu Leu Leu Th #r Ala Ala His Leu Leu            260       #           265       #           270ttt gtg gcg ccg cac aac gac tcg gcc acc gg#g gag ccc gag gcg tcc      864Phe Val Ala Pro His Asn Asp Ser Ala Thr Gl #y Glu Pro Glu Ala Ser        275           #       280           #       285tcg ggc tcg ggg ccg cct tcc ggg ggc gca ct#g ggg cct cgg gcg ctg      912Ser Gly Ser Gly Pro Pro Ser Gly Gly Ala Le #u Gly Pro Arg Ala Leu    290               #   295               #   300ttc gcc agc cgc gtg cgc ccg ggc cag cgc gt#g tac gtg gtg gcc gag      960Phe Ala Ser Arg Val Arg Pro Gly Gln Arg Va #l Tyr Val Val Ala Glu305                 3 #10                 3 #15                 3 #20cgt gac ggg gac cgc cgg ctc ctg ccc gcc gc#t gtg cac agc gtg acc     1008Arg Asp Gly Asp Arg Arg Leu Leu Pro Ala Al #a Val His Ser Val Thr                325   #               330   #               335cta agc gag gag gcc gcg ggc gcc tac gcg cc#g ctc acg gcc cag ggc     1056Leu Ser Glu Glu Ala Ala Gly Ala Tyr Ala Pr #o Leu Thr Ala Gln Gly            340       #           345       #           350acc att ctc atc aac cgg gtg ctg gcc tcg tg#c tac gcg gtc atc gag     1104Thr Ile Leu Ile Asn Arg Val Leu Ala Ser Cy #s Tyr Ala Val Ile Glu        355           #       360           #       365gag cac agc tgg gcg cac cgg gcc ttc gcg cc#c ttc cgc ctg gcg cac     1152Glu His Ser Trp Ala His Arg Ala Phe Ala Pr #o Phe Arg Leu Ala His    370               #   375               #   380gcg ctc ctg gct gca ctg gcg ccc gcg cgc ac#g gac cgc ggc ggg gac     1200Ala Leu Leu Ala Ala Leu Ala Pro Ala Arg Th #r Asp Arg Gly Gly Asp385                 3 #90                 3 #95                 4 #00agc ggc ggc ggg gac cgc ggg ggc ggc ggc gg#c aga gta gcc cta acc     1248Ser Gly Gly Gly Asp Arg Gly Gly Gly Gly Gl #y Arg Val Ala Leu Thr                405   #               410   #               415gct cca ggt gct gcc gac gct ccg ggt gcg gg#g gcc acc gcg ggc atc     1296Ala Pro Gly Ala Ala Asp Ala Pro Gly Ala Gl #y Ala Thr Ala Gly Ile            420       #           425       #           430cac tgg tac tcg cag ctg ctc tac caa ata gg#c acc tgg ctc ctg gac     1344His Trp Tyr Ser Gln Leu Leu Tyr Gln Ile Gl #y Thr Trp Leu Leu Asp        435           #       440           #       445agc gag gcc ctg cac ccg ctg ggc atg gcg gt#c aag tcc agc nnn agc     1392Ser Glu Ala Leu His Pro Leu Gly Met Ala Va #l Lys Ser Ser Xaa Ser    450               #   455               #   460cgg ggg gcc ggg gga ggg gcg cgg gag ggg gc #c                  #       1425 Arg Gly Ala Gly Gly Gly Ala Arg Glu Gly Al #a465                 4 #70                 4 #75 <210> SEQ ID NO 7<211> LENGTH: 1622 <212> TYPE: DNA <213> ORGANISM: Homo sapien Ihh<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (51)..(1283)<400> SEQUENCE: 7catcagccca ccaggagacc tcgcccgccg ctcccccggg ctccccggcc at#g tct        56                    #                  #                   # Met Ser                    #                  #                   #   1 ccc gcc cgg ctc cgg ccc cga ctg cac ttc tg#c ctg gtc ctg ttg ctg      104Pro Ala Arg Leu Arg Pro Arg Leu His Phe Cy #s Leu Val Leu Leu Leu          5         #          10         #          15ctg ctg gtg gtg ccc gcg gca tgg ggc tgc gg#g ccg ggt cgg gtg gtg      152Leu Leu Val Val Pro Ala Ala Trp Gly Cys Gl #y Pro Gly Arg Val Val     20              #     25              #     30ggc agc cgc cgg cga ccg cca cgc aaa ctc gt#g ccg ctc gcc tac aag      200Gly Ser Arg Arg Arg Pro Pro Arg Lys Leu Va #l Pro Leu Ala Tyr Lys 35                  # 40                  # 45                  # 50cag ttc agc ccc aat gtg ccc gag aag acc ct#g ggc gcc agc gga cgc      248Gln Phe Ser Pro Asn Val Pro Glu Lys Thr Le #u Gly Ala Ser Gly Arg                 55  #                 60  #                 65tat gaa ggc aag atc gct cgc agc tcc gag cg#c ttc aag gag ctc acc      296Tyr Glu Gly Lys Ile Ala Arg Ser Ser Glu Ar #g Phe Lys Glu Leu Thr             70      #             75      #             80ccc aat tac aat cca gac atc atc ttc aag ga#c gag gag aac aca ggc      344Pro Asn Tyr Asn Pro Asp Ile Ile Phe Lys As #p Glu Glu Asn Thr Gly         85          #         90          #         95gcc gac cgc ctc atg acc cag cgc tgc aag ga#c cgc ctg aac tcg ctg      392Ala Asp Arg Leu Met Thr Gln Arg Cys Lys As #p Arg Leu Asn Ser Leu    100               #   105               #   110gct atc tcg gtg atg aac cag tgg ccc ggt gt#g aag ctg cgg gtg acc      440Ala Ile Ser Val Met Asn Gln Trp Pro Gly Va #l Lys Leu Arg Val Thr115                 1 #20                 1 #25                 1 #30gag ggc tgg gac gag gac ggc cac cac tca ga#g gag tcc ctg cat tat      488Glu Gly Trp Asp Glu Asp Gly His His Ser Gl #u Glu Ser Leu His Tyr                135   #               140   #               145gag ggc cgc gcg gtg gac atc acc aca tca ga#c cgc gac cgc aat aag      536Glu Gly Arg Ala Val Asp Ile Thr Thr Ser As #p Arg Asp Arg Asn Lys            150       #           155       #           160tat gga ctg ctg gcg cgc ttg gca gtg gag gc#c ggc ttt gac tgg gtg      584Tyr Gly Leu Leu Ala Arg Leu Ala Val Glu Al #a Gly Phe Asp Trp Val        165           #       170           #       175tat tac gag tca aag gcc cac gtg cat tgc tc#c gtc aag tcc gag cac      632Tyr Tyr Glu Ser Lys Ala His Val His Cys Se #r Val Lys Ser Glu His    180               #   185               #   190tcg gcc gca gcc aag acg ggc ggc tgc ttc cc#t gcc gga gcc cag gta      680Ser Ala Ala Ala Lys Thr Gly Gly Cys Phe Pr #o Ala Gly Ala Gln Val195                 2 #00                 2 #05                 2 #10cgc ctg gag agt ggg gcg cgt gtg gcc ttg tc#a gcc gtg agg ccg gga      728Arg Leu Glu Ser Gly Ala Arg Val Ala Leu Se #r Ala Val Arg Pro Gly                215   #               220   #               225gac cgt gtg ctg gcc atg ggg gag gat ggg ag#c ccc acc ttc agc gat      776Asp Arg Val Leu Ala Met Gly Glu Asp Gly Se #r Pro Thr Phe Ser Asp            230       #           235       #           240gtg ctc att ttc ctg gac cgc gag ccc cac ag#g ctg aga gcc ttc cag      824Val Leu Ile Phe Leu Asp Arg Glu Pro His Ar #g Leu Arg Ala Phe Gln        245           #       250           #       255gtc atc gag act cag gac ccc cca cgc cgc ct#g gca ctc aca ccc gct      872Val Ile Glu Thr Gln Asp Pro Pro Arg Arg Le #u Ala Leu Thr Pro Ala    260               #   265               #   270cac ctg ctc ttt acg gct gac aat cac acg ga#g ccg gca gcc cgc ttc      920His Leu Leu Phe Thr Ala Asp Asn His Thr Gl #u Pro Ala Ala Arg Phe275                 2 #80                 2 #85                 2 #90cgg gcc aca ttt gcc agc cac gtg cag cct gg#c cag tac gtg ctg gtg      968Arg Ala Thr Phe Ala Ser His Val Gln Pro Gl #y Gln Tyr Val Leu Val                295   #               300   #               305gct ggg gtg cca ggc ctg cag cct gcc cgc gt#g gca gct gtc tct aca     1016Ala Gly Val Pro Gly Leu Gln Pro Ala Arg Va #l Ala Ala Val Ser Thr            310       #           315       #           320cac gtg gcc ctc ggg gcc tac gcc ccg ctc ac#a aag cat ggg aca ctg     1064His Val Ala Leu Gly Ala Tyr Ala Pro Leu Th #r Lys His Gly Thr Leu        325           #       330           #       335gtg gtg gag gat gtg gtg gca tcc tgc ttc gc#g gcc gtg gct gac cac     1112Val Val Glu Asp Val Val Ala Ser Cys Phe Al #a Ala Val Ala Asp His    340               #   345               #   350cac ctg gct cag ttg gcc ttc tgg ccc ctg ag#a ctc ttt cac agc ttg     1160His Leu Ala Gln Leu Ala Phe Trp Pro Leu Ar #g Leu Phe His Ser Leu355                 3 #60                 3 #65                 3 #70gca tgg ggc agc tgg acc ccg ggg gag ggt gt#g cat tgg tac ccc cag     1208Ala Trp Gly Ser Trp Thr Pro Gly Glu Gly Va #l His Trp Tyr Pro Gln                375   #               380   #               385ctg ctc tac cgc ctg ggg cgt ctc ctg cta ga#a gag ggc agc ttc cac     1256Leu Leu Tyr Arg Leu Gly Arg Leu Leu Leu Gl #u Glu Gly Ser Phe His            390       #           395       #           400cca ctg ggc atg tcc ggg gca ggg agc tgaaaggac#t ccaccgctgc           1303 Pro Leu Gly Met Ser Gly Ala Gly Ser        405           #       410cctcctggaa ctgctgtact gggtccagaa gcctctcagc caggagggag ct#ggccctgg   1363aagggacctg agctggggga cactggctcc tgccatctcc tctgccatga ag#atacacca   1423ttgagacttg actgggcaac accagcgtcc cccacccgcg tcgtggtgta gt#catagagc   1483tgcaagctga gctggcgagg ggatggttgt tgacccctct ctcctagaga cc#ttgaggct   1543ggcacggcga ctcccaactc agcctgctct cactacgagt tttcatactc tg#cctccccc   1603 attgggaggg cccattccc              #                  #                 162 #2 <210> SEQ ID NO 8 <211> LENGTH: 1190<212> TYPE: DNA <213> ORGANISM: Homo sapien Dhh <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(1188) <400> SEQUENCE: 8atg gct ctc ctg acc aat cta ctg ccc ttg tg#c tgc ttg gca ctt ctg       48Met Ala Leu Leu Thr Asn Leu Leu Pro Leu Cy #s Cys Leu Ala Leu Leu  1               5  #                 10  #                 15gcg ctg cca gcc cag agc tgc ggg ccg ggc cg#g ggg ccg gtt ggc cgg       96Ala Leu Pro Ala Gln Ser Cys Gly Pro Gly Ar #g Gly Pro Val Gly Arg             20      #             25      #             30cgc cgc tat gcg cgc aag cag ctc gtg ccg ct#a ctc tac aag caa ttt      144Arg Arg Tyr Ala Arg Lys Gln Leu Val Pro Le #u Leu Tyr Lys Gln Phe         35          #         40          #         45gtg ccc ggc gtg cca gag cgg acc ctg ggc gc#c agt ggg cca gcg gag      192Val Pro Gly Val Pro Glu Arg Thr Leu Gly Al #a Ser Gly Pro Ala Glu     50              #     55              #     60ggg agg gtg gca agg ggc tcc gag cgc ttc cg#g gac ctc gtg ccc aac      240Gly Arg Val Ala Arg Gly Ser Glu Arg Phe Ar #g Asp Leu Val Pro Asn 65                  # 70                  # 75                  # 80tac aac ccc gac atc atc ttc aag gat gag ga#g aac agt gga gcc gac      288Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Gl #u Asn Ser Gly Ala Asp                 85  #                 90  #                 95cgc ctg atg acc gag cgt tgc aag gag agg gt#g aac gct ttg gcc att      336Arg Leu Met Thr Glu Arg Cys Lys Glu Arg Va #l Asn Ala Leu Ala Ile            100       #           105       #           110gcc gtg atg aac atg tgg ccc gga gtg cgc ct#a cga gtg act gag ggc      384Ala Val Met Asn Met Trp Pro Gly Val Arg Le #u Arg Val Thr Glu Gly        115           #       120           #       125tgg gac gag gac ggc cac cac gct cag gat tc#a ctc cac tac gaa ggc      432Trp Asp Glu Asp Gly His His Ala Gln Asp Se #r Leu His Tyr Glu Gly    130               #   135               #   140cgt gct ttg gac atc act acg tct gac cgc ga#c cgc aac aag tat ggg      480Arg Ala Leu Asp Ile Thr Thr Ser Asp Arg As #p Arg Asn Lys Tyr Gly145                 1 #50                 1 #55                 1 #60ttg ctg gcg cgc ctc gca gtg gaa gcc ggc tt#c gac tgg gtc tac tac      528Leu Leu Ala Arg Leu Ala Val Glu Ala Gly Ph #e Asp Trp Val Tyr Tyr                165   #               170   #               175gag tcc cgc aac cac gtc cac gtg tcg gtc aa#a gct gat aac tca ctg      576Glu Ser Arg Asn His Val His Val Ser Val Ly #s Ala Asp Asn Ser Leu            180       #           185       #           190gcg gtc cgg gcg ggc ggc tgc ttt ccg gga aa#t gca act gtg cgc ctg      624Ala Val Arg Ala Gly Gly Cys Phe Pro Gly As #n Ala Thr Val Arg Leu        195           #       200           #       205tgg agc ggc gag cgg aaa ggg ctg cgg gaa ct#g cac cgc gga gac tgg      672Trp Ser Gly Glu Arg Lys Gly Leu Arg Glu Le #u His Arg Gly Asp Trp    210               #   215               #   220gtt ttg gcg gcc gat gcg tca ggc cgg gtg gt#g ccc acg ccg gtg ctg      720Val Leu Ala Ala Asp Ala Ser Gly Arg Val Va #l Pro Thr Pro Val Leu225                 2 #30                 2 #35                 2 #40ctc ttc ctg gac cgg gac ttg cag cgc cgg gc#t tca ttt gtg gct gtg      768Leu Phe Leu Asp Arg Asp Leu Gln Arg Arg Al #a Ser Phe Val Ala Val                245   #               250   #               255gag acc gag tgg cct cca cgc aaa ctg ttg ct#c acg ccc tgg cac ctg      816Glu Thr Glu Trp Pro Pro Arg Lys Leu Leu Le #u Thr Pro Trp His Leu            260       #           265       #           270gtg ttt gcc gct cga ggg ccg gcg ccc gcg cc#a ggc gac ttt gca ccg      864Val Phe Ala Ala Arg Gly Pro Ala Pro Ala Pr #o Gly Asp Phe Ala Pro        275           #       280           #       285gtg ttc gcg cgc cgg cta cgc gct ggg gac tc#g gtg ctg gcg ccc ggc      912Val Phe Ala Arg Arg Leu Arg Ala Gly Asp Se #r Val Leu Ala Pro Gly    290               #   295               #   300ggg gat gcg ctt cgg cca gcg cgc gtg gcc cg#t gtg gcg cgg gag gaa      960Gly Asp Ala Leu Arg Pro Ala Arg Val Ala Ar #g Val Ala Arg Glu Glu305                 3 #10                 3 #15                 3 #20gcc gtg ggc gtg ttc gcg ccg ctc acc gcg ca#c ggg acg ctg ctg gtg     1008Ala Val Gly Val Phe Ala Pro Leu Thr Ala Hi #s Gly Thr Leu Leu Val                325   #               330   #               335aac gat gtc ctg gcc tct tgc tac gcg gtt ct#g gag agt cac cag tgg     1056Asn Asp Val Leu Ala Ser Cys Tyr Ala Val Le #u Glu Ser His Gln Trp            340       #           345       #           350gcg cac cgc gct ttt gcc ccc ttg aga ctg ct#g cac gcg cta ggg gcg     1104Ala His Arg Ala Phe Ala Pro Leu Arg Leu Le #u His Ala Leu Gly Ala        355           #       360           #       365ctg ctc ccc ggc ggg gcc gtc cag ccg act gg#c atg cat tgg tac tct     1152Leu Leu Pro Gly Gly Ala Val Gln Pro Thr Gl #y Met His Trp Tyr Ser    370               #   375               #   380cgg ctc ctc tac cgc tta gcg gag gag cta ct #g ggc tg               #   1190 Arg Leu Leu Tyr Arg Leu Ala Glu Glu Leu Le #u Gly385                 3 #90                 3 #95 <210> SEQ ID NO 9<211> LENGTH: 1251 <212> TYPE: DNA <213> ORGANISM: Zebrafish Thh<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1248)<400> SEQUENCE: 9 atg gac gta agg ctg cat ctg aag caa ttt gc#t tta ctg tgt ttt atc       48Met Asp Val Arg Leu His Leu Lys Gln Phe Al #a Leu Leu Cys Phe Ile  1               5  #                 10  #                 15agc ttg ctt ctg acg cct tgt gga tta gcc tg#t ggt cct ggt aga ggt       96Ser Leu Leu Leu Thr Pro Cys Gly Leu Ala Cy #s Gly Pro Gly Arg Gly             20      #             25      #             30tat gga aaa cga aga cac cca aag aaa tta ac#c ccg ttg gct tac aag      144Tyr Gly Lys Arg Arg His Pro Lys Lys Leu Th #r Pro Leu Ala Tyr Lys         35          #         40          #         45caa ttc atc ccc aac gtt gct gag aaa acg ct#t gga gcc agc ggc aaa      192Gln Phe Ile Pro Asn Val Ala Glu Lys Thr Le #u Gly Ala Ser Gly Lys     50              #     55              #     60tac gaa ggc aaa atc aca agg aat tca gag ag#a ttt aaa gag ctg att      240Tyr Glu Gly Lys Ile Thr Arg Asn Ser Glu Ar #g Phe Lys Glu Leu Ile 65                  # 70                  # 75                  # 80ccg aat tat aat ccc gat atc atc ttt aag ga#c gag gaa aac aca aac      288Pro Asn Tyr Asn Pro Asp Ile Ile Phe Lys As #p Glu Glu Asn Thr Asn                 85  #                 90  #                 95gct gac agg ctg atg acc aag cgc tgt aag ga#c aag tta aat tcg ttg      336Ala Asp Arg Leu Met Thr Lys Arg Cys Lys As #p Lys Leu Asn Ser Leu            100       #           105       #           110gcc ata tcc gtc atg aac cac tgg ccc ggc gt#g aaa ctg cgc gtc act      384Ala Ile Ser Val Met Asn His Trp Pro Gly Va #l Lys Leu Arg Val Thr        115           #       120           #       125gaa ggc tgg gat gag gat ggt cac cat tta ga#a gaa tct ttg cac tat      432Glu Gly Trp Asp Glu Asp Gly His His Leu Gl #u Glu Ser Leu His Tyr    130               #   135               #   140gag gga cgg gca gtg gac atc act acc tca ga#c agg gat aaa agc aag      480Glu Gly Arg Ala Val Asp Ile Thr Thr Ser As #p Arg Asp Lys Ser Lys145                 1 #50                 1 #55                 1 #60tat ggg atg cta tcc agg ctt gca gtg gag gc#a gga ttc gac tgg gtc      528Tyr Gly Met Leu Ser Arg Leu Ala Val Glu Al #a Gly Phe Asp Trp Val                165   #               170   #               175tat tat gaa tct aaa gcc cac ata cac tgc tc#t gtc aaa gca gaa aat      576Tyr Tyr Glu Ser Lys Ala His Ile His Cys Se #r Val Lys Ala Glu Asn            180       #           185       #           190tca gtg gct gct aaa tca gga gga tgt ttt cc#t ggg tct ggg acg gtg      624Ser Val Ala Ala Lys Ser Gly Gly Cys Phe Pr #o Gly Ser Gly Thr Val        195           #       200           #       205aca ctt ggt gat ggg acg agg aaa ccc atc aa#a gat ctt aaa gtg ggc      672Thr Leu Gly Asp Gly Thr Arg Lys Pro Ile Ly #s Asp Leu Lys Val Gly    210               #   215               #   220gac cgg gtt ttg gct gca gac gag aag gga aa#t gtc tta ata agc gac      720Asp Arg Val Leu Ala Ala Asp Glu Lys Gly As #n Val Leu Ile Ser Asp225                 2 #30                 2 #35                 2 #40ttt att atg ttt ata gac cac gat ccg aca ac#g aga agg caa ttc atc      768Phe Ile Met Phe Ile Asp His Asp Pro Thr Th #r Arg Arg Gln Phe Ile                245   #               250   #               255gtc atc gag acg tca gaa cct ttc acc aag ct#c acc ctc act gcc gcg      816Val Ile Glu Thr Ser Glu Pro Phe Thr Lys Le #u Thr Leu Thr Ala Ala            260       #           265       #           270cac cta gtt ttc gtt gga aac tct tca gca gc#t tcg ggt ata aca gca      864His Leu Val Phe Val Gly Asn Ser Ser Ala Al #a Ser Gly Ile Thr Ala        275           #       280           #       285aca ttt gcc agc aac gtg aag cct gga gat ac#a gtt tta gtg tgg gaa      912Thr Phe Ala Ser Asn Val Lys Pro Gly Asp Th #r Val Leu Val Trp Glu    290               #   295               #   300gac aca tgc gag agc ctc aag agc gtt aca gt#g aaa agg att tac act      960Asp Thr Cys Glu Ser Leu Lys Ser Val Thr Va #l Lys Arg Ile Tyr Thr305                 3 #10                 3 #15                 3 #20gag gag cac gag ggc tct ttt gcg cca gtc ac#c gcg cac gga acc ata     1008Glu Glu His Glu Gly Ser Phe Ala Pro Val Th #r Ala His Gly Thr Ile                325   #               330   #               335ata gtg gat cag gtg ttg gca tcg tgc tac gc#g gtc att gag aac cac     1056Ile Val Asp Gln Val Leu Ala Ser Cys Tyr Al #a Val Ile Glu Asn His            340       #           345       #           350aaa tgg gca cat tgg gct ttt gcg ccg gtc ag#g ttg tgt cac aag ctg     1104Lys Trp Ala His Trp Ala Phe Ala Pro Val Ar #g Leu Cys His Lys Leu        355           #       360           #       365atg acg tgg ctt ttt ccg gct cgt gaa tca aa#c gtc aat ttt cag gag     1152Met Thr Trp Leu Phe Pro Ala Arg Glu Ser As #n Val Asn Phe Gln Glu    370               #   375               #   380gat ggt atc cac tgg tac tca aat atg ctg tt#t cac atc ggc tct tgg     1200Asp Gly Ile His Trp Tyr Ser Asn Met Leu Ph #e His Ile Gly Ser Trp385                 3 #90                 3 #95                 4 #00ctg ctg gac aga gac tct ttc cat cca ctc gg#g att tta cac tta agt     1248Leu Leu Asp Arg Asp Ser Phe His Pro Leu Gl #y Ile Leu His Leu Ser                405   #               410   #               415tga                   #                   #                  #           1251 <210> SEQ ID NO 10 <211> LENGTH: 425 <212> TYPE: PRT<213> ORGANISM: chicken Shh <400> SEQUENCE: 10Met Val Glu Met Leu Leu Leu Thr Arg Ile Le #u Leu Val Gly Phe Ile  1               5  #                 10  #                 15Cys Ala Leu Leu Val Ser Ser Gly Leu Thr Cy #s Gly Pro Gly Arg Gly             20      #             25      #             30Ile Gly Lys Arg Arg His Pro Lys Lys Leu Th #r Pro Leu Ala Tyr Lys         35          #         40          #         45Gln Phe Ile Pro Asn Val Ala Glu Lys Thr Le #u Gly Ala Ser Gly Arg     50              #     55              #     60Tyr Glu Gly Lys Ile Thr Arg Asn Ser Glu Ar #g Phe Lys Glu Leu Thr 65                  # 70                  # 75                  # 80Pro Asn Tyr Asn Pro Asp Ile Ile Phe Lys As #p Glu Glu Asn Thr Gly                 85  #                 90  #                 95Ala Asp Arg Leu Met Thr Gln Arg Cys Lys As #p Lys Leu Asn Ala Leu            100       #           105       #           110Ala Ile Ser Val Met Asn Gln Trp Pro Gly Va #l Lys Leu Arg Val Thr        115           #       120           #       125Glu Gly Trp Asp Glu Asp Gly His His Ser Gl #u Glu Ser Leu His Tyr    130               #   135               #   140Glu Gly Arg Ala Val Asp Ile Thr Thr Ser As #p Arg Asp Arg Ser Lys145                 1 #50                 1 #55                 1 #60Tyr Gly Met Leu Ala Arg Leu Ala Val Glu Al #a Gly Phe Asp Trp Val                165   #               170   #               175Tyr Tyr Glu Ser Lys Ala His Ile His Cys Se #r Val Lys Ala Glu Asn            180       #           185       #           190Ser Val Ala Ala Lys Ser Gly Gly Cys Phe Pr #o Gly Ser Ala Thr Val        195           #       200           #       205His Leu Glu His Gly Gly Thr Lys Leu Val Ly #s Asp Leu Ser Pro Gly    210               #   215               #   220Asp Arg Val Leu Ala Ala Asp Ala Asp Gly Ar #g Leu Leu Tyr Ser Asp225                 2 #30                 2 #35                 2 #40Phe Leu Thr Phe Leu Asp Arg Met Asp Ser Se #r Arg Lys Leu Phe Tyr                245   #               250   #               255Val Ile Glu Thr Arg Gln Pro Arg Ala Arg Le #u Leu Leu Thr Ala Ala            260       #           265       #           270His Leu Leu Phe Val Ala Pro Gln His Asn Gl #n Ser Glu Ala Thr Gly        275           #       280           #       285Ser Thr Ser Gly Gln Ala Leu Phe Ala Ser As #n Val Lys Pro Gly Gln    290               #   295               #   300Arg Val Tyr Val Leu Gly Glu Gly Gly Gln Gl #n Leu Leu Pro Ala Ser305                 3 #10                 3 #15                 3 #20Val His Ser Val Ser Leu Arg Glu Glu Ala Se #r Gly Ala Tyr Ala Pro                325   #               330   #               335Leu Thr Ala Gln Gly Thr Ile Leu Ile Asn Ar #g Val Leu Ala Ser Cys            340       #           345       #           350Tyr Ala Val Ile Glu Glu His Ser Trp Ala Hi #s Trp Ala Phe Ala Pro        355           #       360           #       365Phe Arg Leu Ala Gln Gly Leu Leu Ala Ala Le #u Cys Pro Asp Gly Ala    370               #   375               #   380Ile Pro Thr Ala Ala Thr Thr Thr Thr Gly Il #e His Trp Tyr Ser Arg385                 3 #90                 3 #95                 4 #00Leu Leu Tyr Arg Ile Gly Ser Trp Val Leu As #p Gly Asp Ala Leu His                405   #               410   #               415Pro Leu Gly Met Val Ala Pro Ala Ser             420      #           425 <210> SEQ ID NO 11 <211> LENGTH: 396 <212> TYPE: PRT<213> ORGANISM: murine Dhh <400> SEQUENCE: 11Met Ala Leu Pro Ala Ser Leu Leu Pro Leu Cy #s Cys Leu Ala Leu Leu  1               5  #                 10  #                 15Ala Leu Ser Ala Gln Ser Cys Gly Pro Gly Ar #g Gly Pro Val Gly Arg             20      #             25      #             30Arg Arg Tyr Val Arg Lys Gln Leu Val Pro Le #u Leu Tyr Lys Gln Phe         35          #         40          #         45Val Pro Ser Met Pro Glu Arg Thr Leu Gly Al #a Ser Gly Pro Ala Glu     50              #     55              #     60Gly Arg Val Thr Arg Gly Ser Glu Arg Phe Ar #g Asp Leu Val Pro Asn 65                  # 70                  # 75                  # 80Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Gl #u Asn Ser Gly Ala Asp                 85  #                 90  #                 95Arg Leu Met Thr Glu Arg Cys Lys Glu Arg Va #l Asn Ala Leu Ala Ile            100       #           105       #           110Ala Val Met Asn Met Trp Pro Gly Val Arg Le #u Arg Val Thr Glu Gly        115           #       120           #       125Trp Asp Glu Asp Gly His His Ala Gln Asp Se #r Leu His Tyr Glu Gly    130               #   135               #   140Arg Ala Leu Asp Ile Thr Thr Ser Asp Arg As #p Arg Asn Lys Tyr Gly145                 1 #50                 1 #55                 1 #60Leu Leu Ala Arg Leu Ala Val Glu Ala Gly Ph #e Asp Trp Val Tyr Tyr                165   #               170   #               175Glu Ser Arg Asn His Ile His Val Ser Val Ly #s Ala Asp Asn Ser Leu            180       #           185       #           190Ala Val Arg Ala Gly Gly Cys Phe Pro Gly As #n Ala Thr Val Arg Leu        195           #       200           #       205Arg Ser Gly Glu Arg Lys Gly Leu Arg Glu Le #u His Arg Gly Asp Trp    210               #   215               #   220Val Leu Ala Ala Asp Ala Ala Gly Arg Val Va #l Pro Thr Pro Val Leu225                 2 #30                 2 #35                 2 #40Leu Phe Leu Asp Arg Asp Leu Gln Arg Arg Al #a Ser Phe Val Ala Val                245   #               250   #               255Glu Thr Glu Arg Pro Pro Arg Lys Leu Leu Le #u Thr Pro Trp His Leu            260       #           265       #           270Val Phe Ala Ala Arg Gly Pro Ala Pro Ala Pr #o Gly Asp Phe Ala Pro        275           #       280           #       285Val Phe Ala Arg Arg Leu Arg Ala Gly Asp Se #r Val Leu Ala Pro Gly    290               #   295               #   300Gly Asp Ala Leu Gln Pro Ala Arg Val Ala Ar #g Val Ala Arg Glu Glu305                 3 #10                 3 #15                 3 #20Ala Val Gly Val Phe Ala Pro Leu Thr Ala Hi #s Gly Thr Leu Leu Val                325   #               330   #               335Asn Asp Val Leu Ala Ser Cys Tyr Ala Val Le #u Glu Ser His Gln Trp            340       #           345       #           350Ala His Arg Ala Phe Ala Pro Leu Arg Leu Le #u His Ala Leu Gly Ala        355           #       360           #       365Leu Leu Pro Gly Gly Ala Val Gln Pro Thr Gl #y Met His Trp Tyr Ser    370               #   375               #   380Arg Leu Leu Tyr Arg Leu Ala Glu Glu Leu Me #t Gly 385                 3#90                 3 #95 <210> SEQ ID NO 12 <211> LENGTH: 411<212> TYPE: PRT <213> ORGANISM: murine Ihh <400> SEQUENCE: 12Met Ser Pro Ala Trp Leu Arg Pro Arg Leu Ar #g Phe Cys Leu Phe Leu  1               5  #                 10  #                 15Leu Leu Leu Leu Leu Val Pro Ala Ala Arg Gl #y Cys Gly Pro Gly Arg             20      #             25      #             30Val Val Gly Ser Arg Arg Arg Pro Pro Arg Ly #s Leu Val Pro Leu Ala         35          #         40          #         45Tyr Lys Gln Phe Ser Pro Asn Val Pro Glu Ly #s Thr Leu Gly Ala Ser     50              #     55              #     60Gly Arg Tyr Glu Gly Lys Ile Ala Arg Ser Se #r Glu Arg Phe Lys Glu 65                  # 70                  # 75                  # 80Leu Thr Pro Asn Tyr Asn Pro Asp Ile Ile Ph #e Lys Asp Glu Glu Asn                 85  #                 90  #                 95Thr Gly Ala Asp Arg Leu Met Thr Gln Arg Cy #s Lys Asp Arg Leu Asn            100       #           105       #           110Ser Leu Ala Ile Ser Val Met Asn Gln Trp Pr #o Gly Val Lys Leu Arg        115           #       120           #       125Val Thr Glu Gly Arg Asp Glu Asp Gly His Hi #s Ser Glu Glu Ser Leu    130               #   135               #   140His Tyr Glu Gly Arg Ala Val Asp Ile Thr Th #r Ser Asp Arg Asp Arg145                 1 #50                 1 #55                 1 #60Asn Lys Tyr Gly Leu Leu Ala Arg Leu Ala Va #l Glu Ala Gly Phe Asp                165   #               170   #               175Trp Val Tyr Tyr Glu Ser Lys Ala His Val Hi #s Cys Ser Val Lys Ser            180       #           185       #           190Glu His Ser Ala Ala Ala Lys Thr Gly Gly Cy #s Phe Pro Ala Gly Ala        195           #       200           #       205Gln Val Arg Leu Glu Asn Gly Glu Arg Val Al #a Leu Ser Ala Val Lys    210               #   215               #   220Pro Gly Asp Arg Val Leu Ala Met Gly Glu As #p Gly Thr Pro Thr Phe225                 2 #30                 2 #35                 2 #40Ser Asp Val Leu Ile Phe Leu Asp Arg Glu Pr #o Asn Arg Leu Arg Ala                245   #               250   #               255Phe Gln Val Ile Glu Thr Gln Asp Pro Pro Ar #g Arg Leu Ala Leu Thr            260       #           265       #           270Pro Ala His Leu Leu Phe Ile Ala Asp Asn Hi #s Thr Glu Pro Ala Ala        275           #       280           #       285His Phe Arg Ala Thr Phe Ala Ser His Val Gl #n Pro Gly Gln Tyr Val    290               #   295               #   300Leu Val Ser Gly Val Pro Gly Leu Gln Pro Al #a Arg Val Ala Ala Val305                 3 #10                 3 #15                 3 #20Ser Thr His Val Ala Leu Gly Ser Tyr Ala Pr #o Leu Thr Arg His Gly                325   #               330   #               335Thr Leu Val Val Glu Asp Val Val Ala Ser Cy #s Phe Ala Ala Val Ala            340       #           345       #           350Asp His His Leu Ala Gln Leu Ala Phe Trp Pr #o Leu Arg Leu Phe Pro        355           #       360           #       365Ser Leu Ala Trp Gly Ser Trp Thr Pro Ser Gl #u Gly Val His Ser Tyr    370               #   375               #   380Pro Gln Met Leu Tyr Arg Leu Gly Arg Leu Le #u Leu Glu Glu Ser Thr385                 3 #90                 3 #95                 4 #00Phe His Pro Leu Gly Met Ser Gly Ala Gly Se #r                 405  #               410 <210> SEQ ID NO 13 <211> LENGTH: 437 <212> TYPE: PRT<213> ORGANISM: murine Shh <400> SEQUENCE: 13Met Leu Leu Leu Leu Ala Arg Cys Phe Leu Va #l Ile Leu Ala Ser Ser  1               5  #                 10  #                 15Leu Leu Val Cys Pro Gly Leu Ala Cys Gly Pr #o Gly Arg Gly Phe Gly             20      #             25      #             30Lys Arg Arg His Pro Lys Lys Leu Thr Pro Le #u Ala Tyr Lys Gln Phe         35          #         40          #         45Ile Pro Asn Val Ala Glu Lys Thr Leu Gly Al #a Ser Gly Arg Tyr Glu     50              #     55              #     60Gly Lys Ile Thr Arg Asn Ser Glu Arg Phe Ly #s Glu Leu Thr Pro Asn 65                  # 70                  # 75                  # 80Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Gl #u Asn Thr Gly Ala Asp                 85  #                 90  #                 95Arg Leu Met Thr Gln Arg Cys Lys Asp Lys Le #u Asn Ala Leu Ala Ile            100       #           105       #           110Ser Val Met Asn Gln Trp Pro Gly Val Arg Le #u Arg Val Thr Glu Gly        115           #       120           #       125Trp Asp Glu Asp Gly His His Ser Glu Glu Se #r Leu His Tyr Glu Gly    130               #   135               #   140Arg Ala Val Asp Ile Thr Thr Ser Asp Arg As #p Arg Ser Lys Tyr Gly145                 1 #50                 1 #55                 1 #60Met Leu Ala Arg Leu Ala Val Glu Ala Gly Ph #e Asp Trp Val Tyr Tyr                165   #               170   #               175Glu Ser Lys Ala His Ile His Cys Ser Val Ly #s Ala Glu Asn Ser Val            180       #           185       #           190Ala Ala Lys Ser Gly Gly Cys Phe Pro Gly Se #r Ala Thr Val His Leu        195           #       200           #       205Glu Gln Gly Gly Thr Lys Leu Val Lys Asp Le #u Arg Pro Gly Asp Arg    210               #   215               #   220Val Leu Ala Ala Asp Asp Gln Gly Arg Leu Le #u Tyr Ser Asp Phe Leu225                 2 #30                 2 #35                 2 #40Thr Phe Leu Asp Arg Asp Glu Gly Ala Lys Ly #s Val Phe Tyr Val Ile                245   #               250   #               255Glu Thr Leu Glu Pro Arg Glu Arg Leu Leu Le #u Thr Ala Ala His Leu            260       #           265       #           270Leu Phe Val Ala Pro His Asn Asp Ser Gly Pr #o Thr Pro Gly Pro Ser        275           #       280           #       285Ala Leu Phe Ala Ser Arg Val Arg Pro Gly Gl #n Arg Val Tyr Val Val    290               #   295               #   300Ala Glu Arg Gly Gly Asp Arg Arg Leu Leu Pr #o Ala Ala Val His Ser305                 3 #10                 3 #15                 3 #20Val Thr Leu Arg Glu Glu Glu Ala Gly Ala Ty #r Ala Pro Leu Thr Ala                325   #               330   #               335His Gly Thr Ile Leu Ile Asn Arg Val Leu Al #a Ser Cys Tyr Ala Val            340       #           345       #           350Ile Glu Glu His Ser Trp Ala His Arg Ala Ph #e Ala Pro Phe Arg Leu        355           #       360           #       365Ala His Ala Leu Leu Ala Ala Leu Ala Pro Al #a Arg Thr Asp Gly Gly    370               #   375               #   380Gly Gly Gly Ser Ile Pro Ala Ala Gln Ser Al #a Thr Glu Ala Arg Gly385                 3 #90                 3 #95                 4 #00Ala Glu Pro Thr Ala Gly Ile His Trp Tyr Se #r Gln Leu Leu Tyr His                405   #               410   #               415Ile Gly Thr Trp Leu Leu Asp Ser Glu Thr Me #t His Pro Leu Gly Met            420       #           425       #           430Ala Val Lys Ser Ser         435 <210> SEQ ID NO 14 <211> LENGTH: 418<212> TYPE: PRT <213> ORGANISM: zebrafish Shh <400> SEQUENCE: 14Met Arg Leu Leu Thr Arg Val Leu Leu Val Se #r Leu Leu Thr Leu Ser  1               5  #                 10  #                 15Leu Val Val Ser Gly Leu Ala Cys Gly Pro Gl #y Arg Gly Tyr Gly Arg             20      #             25      #             30Arg Arg His Pro Lys Lys Leu Thr Pro Leu Al #a Tyr Lys Gln Phe Ile         35          #         40          #         45Pro Asn Val Ala Glu Lys Thr Leu Gly Ala Se #r Gly Arg Tyr Glu Gly     50              #     55              #     60Lys Ile Thr Arg Asn Ser Glu Arg Phe Lys Gl #u Leu Thr Pro Asn Tyr 65                  # 70                  # 75                  # 80Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu As #n Thr Gly Ala Asp Arg                 85  #                 90  #                 95Leu Met Thr Gln Arg Cys Lys Asp Lys Leu As #n Ser Leu Ala Ile Ser            100       #           105       #           110Val Met Asn His Trp Pro Gly Val Lys Leu Ar #g Val Thr Glu Gly Trp        115           #       120           #       125Asp Glu Asp Gly His His Phe Glu Glu Ser Le #u His Tyr Glu Gly Arg    130               #   135               #   140Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Ly #s Ser Lys Tyr Gly Thr145                 1 #50                 1 #55                 1 #60Leu Ser Arg Leu Ala Val Glu Ala Gly Phe As #p Trp Val Tyr Tyr Glu                165   #               170   #               175Ser Lys Ala His Ile His Cys Ser Val Lys Al #a Glu Asn Ser Val Ala            180       #           185       #           190Ala Lys Ser Gly Gly Cys Phe Pro Gly Ser Al #a Leu Val Ser Leu Gln        195           #       200           #       205Asp Gly Gly Gln Lys Ala Val Lys Asp Leu As #n Pro Gly Asp Lys Val    210               #   215               #   220Leu Ala Ala Asp Ser Ala Gly Asn Leu Val Ph #e Ser Asp Phe Ile Met225                 2 #30                 2 #35                 2 #40Phe Thr Asp Arg Asp Ser Thr Thr Arg Arg Va #l Phe Tyr Val Ile Glu                245   #               250   #               255Thr Gln Glu Pro Val Glu Lys Ile Thr Leu Th #r Ala Ala His Leu Leu            260       #           265       #           270Phe Val Leu Asp Asn Ser Thr Glu Asp Leu Hi #s Thr Met Thr Ala Ala        275           #       280           #       285Tyr Ala Ser Ser Val Arg Ala Gly Gln Lys Va #l Met Val Val Asp Asp    290               #   295               #   300Ser Gly Gln Leu Lys Ser Val Ile Val Gln Ar #g Ile Tyr Thr Glu Glu305                 3 #10                 3 #15                 3 #20Gln Arg Gly Ser Phe Ala Pro Val Thr Ala Hi #s Gly Thr Ile Val Val                325   #               330   #               335Asp Arg Ile Leu Ala Ser Cys Tyr Ala Val Il #e Glu Asp Gln Gly Leu            340       #           345       #           350Ala His Leu Ala Phe Ala Pro Ala Arg Leu Ty #r Tyr Tyr Val Ser Ser        355           #       360           #       365Phe Leu Ser Pro Lys Thr Pro Ala Val Gly Pr #o Met Arg Leu Tyr Asn    370               #   375               #   380Arg Arg Gly Ser Thr Gly Thr Pro Gly Ser Cy #s His Gln Met Gly Thr385                 3 #90                 3 #95                 4 #00Trp Leu Leu Asp Ser Asn Met Leu His Pro Le #u Gly Met Ser Val Asn                405   #               410   #               415 Ser Ser<210> SEQ ID NO 15 <211> LENGTH: 475 <212> TYPE: PRT<213> ORGANISM: Homo sapien Shh <220> FEATURE:<223> OTHER INFORMATION: Xaa at position 463 is # any or unknown amino      acid <400> SEQUENCE: 15 Met Leu Leu Leu Ala Arg Cys Leu Leu Leu Va#l Leu Val Ser Ser Leu   1               5  #                 10 #                 15 Leu Val Cys Ser Gly Leu Ala Cys Gly Pro Gl#y Arg Gly Phe Gly Lys              20      #             25     #             30 Arg Arg His Pro Lys Lys Leu Thr Pro Leu Al#a Tyr Lys Gln Phe Ile          35          #         40         #         45 Pro Asn Val Ala Glu Lys Thr Leu Gly Ala Se#r Gly Arg Tyr Glu Gly      50              #     55             #     60 Lys Ile Ser Arg Asn Ser Glu Arg Phe Lys Gl#u Leu Thr Pro Asn Tyr  65                  # 70                 # 75                  # 80 Asn Pro Asp Ile Ile Phe Lys Asp Glu Glu As#n Thr Gly Ala Asp Arg                  85  #                 90 #                 95 Leu Met Thr Gln Arg Cys Lys Asp Lys Leu As#n Ala Leu Ala Ile Ser             100       #           105      #           110 Val Met Asn Gln Trp Pro Gly Val Lys Leu Ar#g Val Thr Glu Gly Trp         115           #       120          #       125 Asp Glu Asp Gly His His Ser Glu Glu Ser Le#u His Tyr Glu Gly Arg     130               #   135              #   140 Ala Val Asp Ile Thr Thr Ser Asp Arg Asp Ar#g Ser Lys Tyr Gly Met 145                 1 #50                 1#55                 1 #60 Leu Ala Arg Leu Ala Val Glu Ala Gly Phe As#p Trp Val Tyr Tyr Glu                 165   #               170  #               175 Ser Lys Ala His Ile His Cys Ser Val Lys Al#a Glu Asn Ser Val Ala             180       #           185      #           190 Ala Lys Ser Gly Gly Cys Phe Pro Gly Ser Al#a Thr Val His Leu Glu         195           #       200          #       205 Gln Gly Gly Thr Lys Leu Val Lys Asp Leu Se#r Pro Gly Asp Arg Val     210               #   215              #   220 Leu Ala Ala Asp Asp Gln Gly Arg Leu Leu Ty#r Ser Asp Phe Leu Thr 225                 2 #30                 2#35                 2 #40 Phe Leu Asp Arg Asp Asp Gly Ala Lys Lys Va#l Phe Tyr Val Ile Glu                 245   #               250  #               255 Thr Arg Glu Pro Arg Glu Arg Leu Leu Leu Th#r Ala Ala His Leu Leu             260       #           265      #           270 Phe Val Ala Pro His Asn Asp Ser Ala Thr Gl#y Glu Pro Glu Ala Ser         275           #       280          #       285 Ser Gly Ser Gly Pro Pro Ser Gly Gly Ala Le#u Gly Pro Arg Ala Leu     290               #   295              #   300 Phe Ala Ser Arg Val Arg Pro Gly Gln Arg Va#l Tyr Val Val Ala Glu 305                 3 #10                 3#15                 3 #20 Arg Asp Gly Asp Arg Arg Leu Leu Pro Ala Al#a Val His Ser Val Thr                 325   #               330  #               335 Leu Ser Glu Glu Ala Ala Gly Ala Tyr Ala Pr#o Leu Thr Ala Gln Gly             340       #           345      #           350 Thr Ile Leu Ile Asn Arg Val Leu Ala Ser Cy#s Tyr Ala Val Ile Glu         355           #       360          #       365 Glu His Ser Trp Ala His Arg Ala Phe Ala Pr#o Phe Arg Leu Ala His     370               #   375              #   380 Ala Leu Leu Ala Ala Leu Ala Pro Ala Arg Th#r Asp Arg Gly Gly Asp 385                 3 #90                 3#95                 4 #00 Ser Gly Gly Gly Asp Arg Gly Gly Gly Gly Gl#y Arg Val Ala Leu Thr                 405   #               410  #               415 Ala Pro Gly Ala Ala Asp Ala Pro Gly Ala Gl#y Ala Thr Ala Gly Ile             420       #           425      #           430 His Trp Tyr Ser Gln Leu Leu Tyr Gln Ile Gl#y Thr Trp Leu Leu Asp         435           #       440          #       445 Ser Glu Ala Leu His Pro Leu Gly Met Ala Va#l Lys Ser Ser Xaa Ser     450               #   455              #   460 Arg Gly Ala Gly Gly Gly Ala Arg Glu Gly Al #a465                 4 #70                 4 #75 <210> SEQ ID NO 16<211> LENGTH: 411 <212> TYPE: PRT <213> ORGANISM: Homo sapien Ihh<400> SEQUENCE: 16 Met Ser Pro Ala Arg Leu Arg Pro Arg Leu Hi#s Phe Cys Leu Val Leu   1               5  #                 10 #                 15 Leu Leu Leu Leu Val Val Pro Ala Ala Trp Gl#y Cys Gly Pro Gly Arg              20      #             25     #             30 Val Val Gly Ser Arg Arg Arg Pro Pro Arg Ly#s Leu Val Pro Leu Ala          35          #         40         #         45 Tyr Lys Gln Phe Ser Pro Asn Val Pro Glu Ly#s Thr Leu Gly Ala Ser      50              #     55             #     60 Gly Arg Tyr Glu Gly Lys Ile Ala Arg Ser Se#r Glu Arg Phe Lys Glu  65                  # 70                 # 75                  # 80 Leu Thr Pro Asn Tyr Asn Pro Asp Ile Ile Ph#e Lys Asp Glu Glu Asn                  85  #                 90 #                 95 Thr Gly Ala Asp Arg Leu Met Thr Gln Arg Cy#s Lys Asp Arg Leu Asn             100       #           105      #           110 Ser Leu Ala Ile Ser Val Met Asn Gln Trp Pr#o Gly Val Lys Leu Arg         115           #       120          #       125 Val Thr Glu Gly Trp Asp Glu Asp Gly His Hi#s Ser Glu Glu Ser Leu     130               #   135              #   140 His Tyr Glu Gly Arg Ala Val Asp Ile Thr Th#r Ser Asp Arg Asp Arg 145                 1 #50                 1#55                 1 #60 Asn Lys Tyr Gly Leu Leu Ala Arg Leu Ala Va#l Glu Ala Gly Phe Asp                 165   #               170  #               175 Trp Val Tyr Tyr Glu Ser Lys Ala His Val Hi#s Cys Ser Val Lys Ser             180       #           185      #           190 Glu His Ser Ala Ala Ala Lys Thr Gly Gly Cy#s Phe Pro Ala Gly Ala         195           #       200          #       205 Gln Val Arg Leu Glu Ser Gly Ala Arg Val Al#a Leu Ser Ala Val Arg     210               #   215              #   220 Pro Gly Asp Arg Val Leu Ala Met Gly Glu As#p Gly Ser Pro Thr Phe 225                 2 #30                 2#35                 2 #40 Ser Asp Val Leu Ile Phe Leu Asp Arg Glu Pr#o His Arg Leu Arg Ala                 245   #               250  #               255 Phe Gln Val Ile Glu Thr Gln Asp Pro Pro Ar#g Arg Leu Ala Leu Thr             260       #           265      #           270 Pro Ala His Leu Leu Phe Thr Ala Asp Asn Hi#s Thr Glu Pro Ala Ala         275           #       280          #       285 Arg Phe Arg Ala Thr Phe Ala Ser His Val Gl#n Pro Gly Gln Tyr Val     290               #   295              #   300 Leu Val Ala Gly Val Pro Gly Leu Gln Pro Al#a Arg Val Ala Ala Val 305                 3 #10                 3#15                 3 #20 Ser Thr His Val Ala Leu Gly Ala Tyr Ala Pr#o Leu Thr Lys His Gly                 325   #               330  #               335 Thr Leu Val Val Glu Asp Val Val Ala Ser Cy#s Phe Ala Ala Val Ala             340       #           345      #           350 Asp His His Leu Ala Gln Leu Ala Phe Trp Pr#o Leu Arg Leu Phe His         355           #       360          #       365 Ser Leu Ala Trp Gly Ser Trp Thr Pro Gly Gl#u Gly Val His Trp Tyr     370               #   375              #   380 Pro Gln Leu Leu Tyr Arg Leu Gly Arg Leu Le#u Leu Glu Glu Gly Ser 385                 3 #90                 3#95                 4 #00 Phe His Pro Leu Gly Met Ser Gly Ala Gly Se #r                405   #               410 <210> SEQ ID NO 17<211> LENGTH: 396 <212> TYPE: PRT <213> ORGANISM: Homo sapien Dhh<400> SEQUENCE: 17 Met Ala Leu Leu Thr Asn Leu Leu Pro Leu Cy#s Cys Leu Ala Leu Leu   1               5  #                 10 #                 15 Ala Leu Pro Ala Gln Ser Cys Gly Pro Gly Ar#g Gly Pro Val Gly Arg              20      #             25     #             30 Arg Arg Tyr Ala Arg Lys Gln Leu Val Pro Le#u Leu Tyr Lys Gln Phe          35          #         40         #         45 Val Pro Gly Val Pro Glu Arg Thr Leu Gly Al#a Ser Gly Pro Ala Glu      50              #     55             #     60 Gly Arg Val Ala Arg Gly Ser Glu Arg Phe Ar#g Asp Leu Val Pro Asn  65                  # 70                 # 75                  # 80 Tyr Asn Pro Asp Ile Ile Phe Lys Asp Glu Gl#u Asn Ser Gly Ala Asp                  85  #                 90 #                 95 Arg Leu Met Thr Glu Arg Cys Lys Glu Arg Va#l Asn Ala Leu Ala Ile             100       #           105      #           110 Ala Val Met Asn Met Trp Pro Gly Val Arg Le#u Arg Val Thr Glu Gly         115           #       120          #       125 Trp Asp Glu Asp Gly His His Ala Gln Asp Se#r Leu His Tyr Glu Gly     130               #   135              #   140 Arg Ala Leu Asp Ile Thr Thr Ser Asp Arg As#p Arg Asn Lys Tyr Gly 145                 1 #50                 1#55                 1 #60 Leu Leu Ala Arg Leu Ala Val Glu Ala Gly Ph#e Asp Trp Val Tyr Tyr                 165   #               170  #               175 Glu Ser Arg Asn His Val His Val Ser Val Ly#s Ala Asp Asn Ser Leu             180       #           185      #           190 Ala Val Arg Ala Gly Gly Cys Phe Pro Gly As#n Ala Thr Val Arg Leu         195           #       200          #       205 Trp Ser Gly Glu Arg Lys Gly Leu Arg Glu Le#u His Arg Gly Asp Trp     210               #   215              #   220 Val Leu Ala Ala Asp Ala Ser Gly Arg Val Va#l Pro Thr Pro Val Leu 225                 2 #30                 2#35                 2 #40 Leu Phe Leu Asp Arg Asp Leu Gln Arg Arg Al#a Ser Phe Val Ala Val                 245   #               250  #               255 Glu Thr Glu Trp Pro Pro Arg Lys Leu Leu Le#u Thr Pro Trp His Leu             260       #           265      #           270 Val Phe Ala Ala Arg Gly Pro Ala Pro Ala Pr#o Gly Asp Phe Ala Pro         275           #       280          #       285 Val Phe Ala Arg Arg Leu Arg Ala Gly Asp Se#r Val Leu Ala Pro Gly     290               #   295              #   300 Gly Asp Ala Leu Arg Pro Ala Arg Val Ala Ar#g Val Ala Arg Glu Glu 305                 3 #10                 3#15                 3 #20 Ala Val Gly Val Phe Ala Pro Leu Thr Ala Hi#s Gly Thr Leu Leu Val                 325   #               330  #               335 Asn Asp Val Leu Ala Ser Cys Tyr Ala Val Le#u Glu Ser His Gln Trp             340       #           345      #           350 Ala His Arg Ala Phe Ala Pro Leu Arg Leu Le#u His Ala Leu Gly Ala         355           #       360          #       365 Leu Leu Pro Gly Gly Ala Val Gln Pro Thr Gl#y Met His Trp Tyr Ser     370               #   375              #   380 Arg Leu Leu Tyr Arg Leu Ala Glu Glu Leu Le #u Gly385                 3 #90                 3 #95 <210> SEQ ID NO 18<211> LENGTH: 416 <212> TYPE: PRT <213> ORGANISM: Zebrafish Thh<400> SEQUENCE: 18 Met Asp Val Arg Leu His Leu Lys Gln Phe Al#a Leu Leu Cys Phe Ile   1               5  #                 10 #                 15 Ser Leu Leu Leu Thr Pro Cys Gly Leu Ala Cy#s Gly Pro Gly Arg Gly              20      #             25     #             30 Tyr Gly Lys Arg Arg His Pro Lys Lys Leu Th#r Pro Leu Ala Tyr Lys          35          #         40         #         45 Gln Phe Ile Pro Asn Val Ala Glu Lys Thr Le#u Gly Ala Ser Gly Lys      50              #     55             #     60 Tyr Glu Gly Lys Ile Thr Arg Asn Ser Glu Ar#g Phe Lys Glu Leu Ile  65                  # 70                 # 75                  # 80 Pro Asn Tyr Asn Pro Asp Ile Ile Phe Lys As#p Glu Glu Asn Thr Asn                  85  #                 90 #                 95 Ala Asp Arg Leu Met Thr Lys Arg Cys Lys As#p Lys Leu Asn Ser Leu             100       #           105      #           110 Ala Ile Ser Val Met Asn His Trp Pro Gly Va#l Lys Leu Arg Val Thr         115           #       120          #       125 Glu Gly Trp Asp Glu Asp Gly His His Leu Gl#u Glu Ser Leu His Tyr     130               #   135              #   140 Glu Gly Arg Ala Val Asp Ile Thr Thr Ser As#p Arg Asp Lys Ser Lys 145                 1 #50                 1#55                 1 #60 Tyr Gly Met Leu Ser Arg Leu Ala Val Glu Al#a Gly Phe Asp Trp Val                 165   #               170  #               175 Tyr Tyr Glu Ser Lys Ala His Ile His Cys Se#r Val Lys Ala Glu Asn             180       #           185      #           190 Ser Val Ala Ala Lys Ser Gly Gly Cys Phe Pr#o Gly Ser Gly Thr Val         195           #       200          #       205 Thr Leu Gly Asp Gly Thr Arg Lys Pro Ile Ly#s Asp Leu Lys Val Gly     210               #   215              #   220 Asp Arg Val Leu Ala Ala Asp Glu Lys Gly As#n Val Leu Ile Ser Asp 225                 2 #30                 2#35                 2 #40 Phe Ile Met Phe Ile Asp His Asp Pro Thr Th#r Arg Arg Gln Phe Ile                 245   #               250  #               255 Val Ile Glu Thr Ser Glu Pro Phe Thr Lys Le#u Thr Leu Thr Ala Ala             260       #           265      #           270 His Leu Val Phe Val Gly Asn Ser Ser Ala Al#a Ser Gly Ile Thr Ala         275           #       280          #       285 Thr Phe Ala Ser Asn Val Lys Pro Gly Asp Th#r Val Leu Val Trp Glu     290               #   295              #   300 Asp Thr Cys Glu Ser Leu Lys Ser Val Thr Va#l Lys Arg Ile Tyr Thr 305                 3 #10                 3#15                 3 #20 Glu Glu His Glu Gly Ser Phe Ala Pro Val Th#r Ala His Gly Thr Ile                 325   #               330  #               335 Ile Val Asp Gln Val Leu Ala Ser Cys Tyr Al#a Val Ile Glu Asn His             340       #           345      #           350 Lys Trp Ala His Trp Ala Phe Ala Pro Val Ar#g Leu Cys His Lys Leu         355           #       360          #       365 Met Thr Trp Leu Phe Pro Ala Arg Glu Ser As#n Val Asn Phe Gln Glu     370               #   375              #   380 Asp Gly Ile His Trp Tyr Ser Asn Met Leu Ph#e His Ile Gly Ser Trp 385                 3 #90                 3#95                 4 #00 Leu Leu Asp Arg Asp Ser Phe His Pro Leu Gl#y Ile Leu His Leu Ser                 405   #               410  #               415 <210> SEQ ID NO 19 <211> LENGTH: 1416<212> TYPE: DNA <213> ORGANISM: Drosophila HH <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(1413) <400> SEQUENCE: 19atg gat aac cac agc tca gtg cct tgg gcc ag#t gcc gcc agt gtc acc       48Met Asp Asn His Ser Ser Val Pro Trp Ala Se #r Ala Ala Ser Val Thr  1               5  #                 10  #                 15tgt ctc tcc ctg gga tgc caa atg cca cag tt#c cag ttc cag ttc cag       96Cys Leu Ser Leu Gly Cys Gln Met Pro Gln Ph #e Gln Phe Gln Phe Gln             20      #             25      #             30ctc caa atc cgc agc gag ctc cat ctc cgc aa#g ccc gca aga aga acg      144Leu Gln Ile Arg Ser Glu Leu His Leu Arg Ly #s Pro Ala Arg Arg Thr         35          #         40          #         45caa acg atg cgc cac att gcg cat acg cag cg#t tgc ctc agc agg ctg      192Gln Thr Met Arg His Ile Ala His Thr Gln Ar #g Cys Leu Ser Arg Leu     50              #     55              #     60acc tct ctg gtg gcc ctg ctg ctg atc gtc tt#g ccg atg gtc ttt agc      240Thr Ser Leu Val Ala Leu Leu Leu Ile Val Le #u Pro Met Val Phe Ser 65                  # 70                  # 75                  # 80ccg gct cac agc tgc ggt cct ggc cga gga tt#g ggt cgt cat agg gcg      288Pro Ala His Ser Cys Gly Pro Gly Arg Gly Le #u Gly Arg His Arg Ala                 85  #                 90  #                 95cgc aac ctg tat ccg ctg gtc ctc aag cag ac#a att ccc aat cta tcc      336Arg Asn Leu Tyr Pro Leu Val Leu Lys Gln Th #r Ile Pro Asn Leu Ser            100       #           105       #           110gag tac acg aac agc gcc tcc gga cct ctg ga#g ggt gtg atc cgt cgg      384Glu Tyr Thr Asn Ser Ala Ser Gly Pro Leu Gl #u Gly Val Ile Arg Arg        115           #       120           #       125gat tcg ccc aaa ttc aag gac ctc gtg ccc aa#c tac aac agg gac atc      432Asp Ser Pro Lys Phe Lys Asp Leu Val Pro As #n Tyr Asn Arg Asp Ile    130               #   135               #   140ctt ttc cgt gac gag gaa ggc acc gga gcg ga#t ggc ttg atg agc aag      480Leu Phe Arg Asp Glu Glu Gly Thr Gly Ala As #p Gly Leu Met Ser Lys145                 1 #50                 1 #55                 1 #60cgc tgc aag gag aag cta aac gtg ctg gcc ta#c tcg gtg atg aac gaa      528Arg Cys Lys Glu Lys Leu Asn Val Leu Ala Ty #r Ser Val Met Asn Glu                165   #               170   #               175tgg ccc ggc atc cgg ctg ctg gtc acc gag ag#c tgg gac gag gac tac      576Trp Pro Gly Ile Arg Leu Leu Val Thr Glu Se #r Trp Asp Glu Asp Tyr            180       #           185       #           190cat cac ggc cag gag tcg ctc cac tac gag gg#c cga gcg gtg acc att      624His His Gly Gln Glu Ser Leu His Tyr Glu Gl #y Arg Ala Val Thr Ile        195           #       200           #       205gcc acc tcc gat cgc gac cag tcc aaa tac gg#c atg ctc gct cgc ctg      672Ala Thr Ser Asp Arg Asp Gln Ser Lys Tyr Gl #y Met Leu Ala Arg Leu    210               #   215               #   220gcc gtc gag gct gga ttc gat tgg gtc tcc ta#c gtc agc agg cgc cac      720Ala Val Glu Ala Gly Phe Asp Trp Val Ser Ty #r Val Ser Arg Arg His225                 2 #30                 2 #35                 2 #40atc tac tgc tcc gtc aag tca gat tcg tcg at#c agt tcc cac gtg cac      768Ile Tyr Cys Ser Val Lys Ser Asp Ser Ser Il #e Ser Ser His Val His                245   #               250   #               255ggc tgc ttc acg ccg gag agc aca gcg ctg ct#g gag agt gga gtc cgg      816Gly Cys Phe Thr Pro Glu Ser Thr Ala Leu Le #u Glu Ser Gly Val Arg            260       #           265       #           270aag ccg ctc ggc gag ctc tct atc gga gat cg#t gtt ttg agc atg acc      864Lys Pro Leu Gly Glu Leu Ser Ile Gly Asp Ar #g Val Leu Ser Met Thr        275           #       280           #       285gcc aac gga cag gcc gtc tac agc gaa gtg at#c ctc ttc atg gac cgc      912Ala Asn Gly Gln Ala Val Tyr Ser Glu Val Il #e Leu Phe Met Asp Arg    290               #   295               #   300aac ctc gag cag atg caa aac ttt gtg cag ct#g cac acg gac ggt gga      960Asn Leu Glu Gln Met Gln Asn Phe Val Gln Le #u His Thr Asp Gly Gly305                 3 #10                 3 #15                 3 #20gca gtg ctc acg gtg acg ccg gct cac ctg gt#t agc gtt tgg cag ccg     1008Ala Val Leu Thr Val Thr Pro Ala His Leu Va #l Ser Val Trp Gln Pro                325   #               330   #               335gag agc cag aag ctc acg ttt gtg ttt gcg ca#t cgc atc gag gag aag     1056Glu Ser Gln Lys Leu Thr Phe Val Phe Ala Hi #s Arg Ile Glu Glu Lys            340       #           345       #           350aac cag gtg ctc gta cgg gat gtg gag acg gg#c gag ctg agg ccc cag     1104Asn Gln Val Leu Val Arg Asp Val Glu Thr Gl #y Glu Leu Arg Pro Gln        355           #       360           #       365cga gtg gtc aag ttg ggc agt gtg cgc agt aa#g ggc gtg gtc gcg ccg     1152Arg Val Val Lys Leu Gly Ser Val Arg Ser Ly #s Gly Val Val Ala Pro    370               #   375               #   380ctg acc cgc gag ggc acc att gtg gtc aac tc#g gtg gcc gcc agt tgc     1200Leu Thr Arg Glu Gly Thr Ile Val Val Asn Se #r Val Ala Ala Ser Cys385                 3 #90                 3 #95                 4 #00tat gcg gtg atc aac agt cag tcg ctg gcc ca#c tgg gga ctg gct ccc     1248Tyr Ala Val Ile Asn Ser Gln Ser Leu Ala Hi #s Trp Gly Leu Ala Pro                405   #               410   #               415atg cgc ctg ctg tcc acg ctg gag gcg tgg ct#g ccc gcc aag gag cag     1296Met Arg Leu Leu Ser Thr Leu Glu Ala Trp Le #u Pro Ala Lys Glu Gln            420       #           425       #           430ttg cac agt tcg ccg aag gtg gtg agc tcg gc#g cag cag cag aat ggc     1344Leu His Ser Ser Pro Lys Val Val Ser Ser Al #a Gln Gln Gln Asn Gly        435           #       440           #       445atc cat tgg tat gcc aat gcg ctc tac aag gt#c aag gac tac gtg ctg     1392Ile His Trp Tyr Ala Asn Ala Leu Tyr Lys Va #l Lys Asp Tyr Val Leu    450               #   455               #   460ccg cag agc tgg cgc cac gat tga      #                  #              1416 Pro Gln Ser Trp Arg His Asp 465                 4#70 <210> SEQ ID NO 20 <211> LENGTH: 471 <212> TYPE: PRT<213> ORGANISM: Drosophila HH <400> SEQUENCE: 20Met Asp Asn His Ser Ser Val Pro Trp Ala Se #r Ala Ala Ser Val Thr  1               5  #                 10  #                 15Cys Leu Ser Leu Gly Cys Gln Met Pro Gln Ph #e Gln Phe Gln Phe Gln             20      #             25      #             30Leu Gln Ile Arg Ser Glu Leu His Leu Arg Ly #s Pro Ala Arg Arg Thr         35          #         40          #         45Gln Thr Met Arg His Ile Ala His Thr Gln Ar #g Cys Leu Ser Arg Leu     50              #     55              #     60Thr Ser Leu Val Ala Leu Leu Leu Ile Val Le #u Pro Met Val Phe Ser 65                  # 70                  # 75                  # 80Pro Ala His Ser Cys Gly Pro Gly Arg Gly Le #u Gly Arg His Arg Ala                 85  #                 90  #                 95Arg Asn Leu Tyr Pro Leu Val Leu Lys Gln Th #r Ile Pro Asn Leu Ser            100       #           105       #           110Glu Tyr Thr Asn Ser Ala Ser Gly Pro Leu Gl #u Gly Val Ile Arg Arg        115           #       120           #       125Asp Ser Pro Lys Phe Lys Asp Leu Val Pro As #n Tyr Asn Arg Asp Ile    130               #   135               #   140Leu Phe Arg Asp Glu Glu Gly Thr Gly Ala As #p Gly Leu Met Ser Lys145                 1 #50                 1 #55                 1 #60Arg Cys Lys Glu Lys Leu Asn Val Leu Ala Ty #r Ser Val Met Asn Glu                165   #               170   #               175Trp Pro Gly Ile Arg Leu Leu Val Thr Glu Se #r Trp Asp Glu Asp Tyr            180       #           185       #           190His His Gly Gln Glu Ser Leu His Tyr Glu Gl #y Arg Ala Val Thr Ile        195           #       200           #       205Ala Thr Ser Asp Arg Asp Gln Ser Lys Tyr Gl #y Met Leu Ala Arg Leu    210               #   215               #   220Ala Val Glu Ala Gly Phe Asp Trp Val Ser Ty #r Val Ser Arg Arg His225                 2 #30                 2 #35                 2 #40Ile Tyr Cys Ser Val Lys Ser Asp Ser Ser Il #e Ser Ser His Val His                245   #               250   #               255Gly Cys Phe Thr Pro Glu Ser Thr Ala Leu Le #u Glu Ser Gly Val Arg            260       #           265       #           270Lys Pro Leu Gly Glu Leu Ser Ile Gly Asp Ar #g Val Leu Ser Met Thr        275           #       280           #       285Ala Asn Gly Gln Ala Val Tyr Ser Glu Val Il #e Leu Phe Met Asp Arg    290               #   295               #   300Asn Leu Glu Gln Met Gln Asn Phe Val Gln Le #u His Thr Asp Gly Gly305                 3 #10                 3 #15                 3 #20Ala Val Leu Thr Val Thr Pro Ala His Leu Va #l Ser Val Trp Gln Pro                325   #               330   #               335Glu Ser Gln Lys Leu Thr Phe Val Phe Ala Hi #s Arg Ile Glu Glu Lys            340       #           345       #           350Asn Gln Val Leu Val Arg Asp Val Glu Thr Gl #y Glu Leu Arg Pro Gln        355           #       360           #       365Arg Val Val Lys Leu Gly Ser Val Arg Ser Ly #s Gly Val Val Ala Pro    370               #   375               #   380Leu Thr Arg Glu Gly Thr Ile Val Val Asn Se #r Val Ala Ala Ser Cys385                 3 #90                 3 #95                 4 #00Tyr Ala Val Ile Asn Ser Gln Ser Leu Ala Hi #s Trp Gly Leu Ala Pro                405   #               410   #               415Met Arg Leu Leu Ser Thr Leu Glu Ala Trp Le #u Pro Ala Lys Glu Gln            420       #           425       #           430Leu His Ser Ser Pro Lys Val Val Ser Ser Al #a Gln Gln Gln Asn Gly        435           #       440           #       445Ile His Trp Tyr Ala Asn Ala Leu Tyr Lys Va #l Lys Asp Tyr Val Leu    450               #   455               #   460Pro Gln Ser Trp Arg His Asp 465                 4 #70 <210> SEQ ID NO 21<211> LENGTH: 221 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: degenerate       polypeptide sequence <220> FEATURE:<222> LOCATION: 7 <223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il#e, Phe, Tyr or Trp <220> FEATURE: <222> LOCATION: 9<223> OTHER INFORMATION: Arg, His or Lys <220> FEATURE:<222> LOCATION: 44 <223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il#e, Ser or Thr <220> FEATURE: <222> LOCATION: 85<223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il #e, Ser or Thr<220> FEATURE: <222> LOCATION: 93<223> OTHER INFORMATION: Lys, Arg, His, Asn or  #Gln <220> FEATURE:<222> LOCATION: 98 <223> OTHER INFORMATION: Lys, Arg or His<220> FEATURE: <222> LOCATION: 112<223> OTHER INFORMATION: Ser, Thr, Tyr, Trp or  #Phe <220> FEATURE:<222> LOCATION: 132 <223> OTHER INFORMATION: Lys, Arg or His<220> FEATURE: <222> LOCATION: 137<223> OTHER INFORMATION: Met, Cys, Ser or Thr <220> FEATURE:<222> LOCATION: 139 <223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il#e, Ser or Thr <220> FEATURE: <222> LOCATION: 181<223> OTHER INFORMATION: Leu, Val, Met, Thr or  #Ser <220> FEATURE:<222> LOCATION: 183 <223> OTHER INFORMATION: His, Phe, Tyr, Ser, Th#r, Met or Cys <220> FEATURE: <222> LOCATION: 185<223> OTHER INFORMATION: Gln, Asn, Glu, or Asp <220> FEATURE:<222> LOCATION: 186 <223> OTHER INFORMATION: His, Phe, Tyr, Thr, Gl#n, Asn, Glu or Asp <220> FEATURE: <222> LOCATION: 189<223> OTHER INFORMATION: Gln, Asn, Glu, Asp, Th #r, Ser, Met or Cys<220> FEATURE: <222> LOCATION: 191<223> OTHER INFORMATION: Ala, Gly, Cys, Leu, Va #l or Met <220> FEATURE:<222> LOCATION: 196 <223> OTHER INFORMATION: Arg, Lys, Met, Ile, As#n, Asp, Glu, Gln, Ser,       Thr or Cys <220> FEATURE:<222> LOCATION: 200 <223> OTHER INFORMATION: Arg, Lys, Met or Ile<220> FEATURE: <222> LOCATION: 206<223> OTHER INFORMATION: Ala, Gly, Cys, Asp, Gl#u, Gln, Asn, Ser, Thr or       Met <220> FEATURE: <222> LOCATION: 207<223> OTHER INFORMATION: Ala, Gly, Cys, Asp, As #n, Glu or Gln<220> FEATURE: <222> LOCATION: 209<223> OTHER INFORMATION: Arg, Lys, Met, Ile, As #n, Asp, Glu or Gln<220> FEATURE: <222> LOCATION: 211<223> OTHER INFORMATION: Leu, Val, Met or Ile <220> FEATURE:<222> LOCATION: 212 <223> OTHER INFORMATION: Phe, Tyr, Thr, His or  #Trp<220> FEATURE: <222> LOCATION: 216<223> OTHER INFORMATION: Ile, Val, Leu or Met <220> FEATURE:<222> LOCATION: 217 <223> OTHER INFORMATION: Met, Cys, Ile, Leu, Va#l, Thr or Ser <220> FEATURE: <222> LOCATION: 219<223> OTHER INFORMATION: Leu, Val, Met, Thr or  #Ser <220> FEATURE:<223> OTHER INFORMATION: each Xaa may also be  #any amino acid.<400> SEQUENCE: 21 Cys Gly Pro Gly Arg Gly Xaa Gly Xaa Arg Ar#g His Pro Lys Lys Leu   1               5  #                 10 #                 15 Thr Pro Leu Ala Tyr Lys Gln Phe Ile Pro As#n Val Ala Glu Lys Thr              20      #             25     #             30 Leu Gly Ala Ser Gly Arg Tyr Glu Gly Lys Il#e Xaa Arg Asn Ser Glu          35          #         40         #         45 Arg Phe Lys Glu Leu Thr Pro Asn Tyr Asn Pr#o Asp Ile Ile Phe Lys      50              #     55             #     60 Asp Glu Glu Asn Thr Gly Ala Asp Arg Leu Me#t Thr Gln Arg Cys Lys  65                  # 70                 # 75                  # 80 Asp Lys Leu Asn Xaa Leu Ala Ile Ser Val Me#t Asn Xaa Trp Pro Gly                  85  #                 90 #                 95 Val Xaa Leu Arg Val Thr Glu Gly Trp Asp Gl#u Asp Gly His His Xaa             100       #           105      #           110 Glu Glu Ser Leu His Tyr Glu Gly Arg Ala Va#l Asp Ile Thr Thr Ser         115           #       120          #       125 Asp Arg Asp Xaa Ser Lys Tyr Gly Xaa Leu Xa#a Arg Leu Ala Val Glu     130               #   135              #   140 Ala Gly Phe Asp Trp Val Tyr Tyr Glu Ser Ly#s Ala His Ile His Cys 145                 1 #50                 1#55                 1 #60 Ser Val Lys Ala Glu Asn Ser Val Ala Ala Ly#s Ser Gly Gly Cys Phe                 165   #               170  #               175 Pro Gly Ser Ala Xaa Val Xaa Leu Xaa Xaa Gl#y Gly Xaa Lys Xaa Val             180       #           185      #           190 Lys Asp Leu Xaa Pro Gly Asp Xaa Val Leu Al#a Ala Asp Xaa Xaa Gly         195           #       200          #       205 Xaa Leu Xaa Xaa Ser Asp Phe Xaa Xaa Phe Xa #a Asp Arg    210               #   215               #   220 <210> SEQ ID NO 22<211> LENGTH: 167 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: degenerate       polypeptide sequence <220> FEATURE:<222> LOCATION: 7 <223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il#e, Pro, Phe or Tyr <220> FEATURE: <222> LOCATION: 8<223> OTHER INFORMATION: Gly, Ala, Val, Leu or  #Ile <220> FEATURE:<222> LOCATION: 9 <223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il#e, Lys, His or Arg <220> FEATURE: <222> LOCATION: 12<223> OTHER INFORMATION: Lys, Arg or His <220> FEATURE:<222> LOCATION: 13 <223> OTHER INFORMATION: Phe, Trp, Tyr or an #amino acid gap <220> FEATURE: <222> LOCATION: 14<223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il #e or an amino acid gap<220> FEATURE: <222> LOCATION: 17<223> OTHER INFORMATION: Asn, Gln, His, Arg or  #Lys <220> FEATURE:<222> LOCATION: 19 <223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il#e, Ser or Thr <220> FEATURE: <222> LOCATION: 22<223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il #e, Ser or Thr<220> FEATURE: <222> LOCATION: 27<223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il #e, Ser or Thr<220> FEATURE: <222> LOCATION: 29<223> OTHER INFORMATION: Ser, Thr, Gln or Asn <220> FEATURE:<222> LOCATION: 30 <223> OTHER INFORMATION: Met, Cys, Gly, Ala, Va#l, Leu, Ile, Ser or Thr <220> FEATURE: <222> LOCATION: 31<223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il #e or Pro <220> FEATURE:<222> LOCATION: 33 <223> OTHER INFORMATION: Arg, His or Lys<220> FEATURE: <222> LOCATION: 40<223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il #e, Pro, Arg, His or Lys<220> FEATURE: <222> LOCATION: 41<223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il #e, Phe or Tyr<220> FEATURE: <222> LOCATION: 44<223> OTHER INFORMATION: Arg, His or Lys <220> FEATURE:<222> LOCATION: 45 <223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il#e, Ser or Thr <220> FEATURE: <222> LOCATION: 46<223> OTHER INFORMATION: Thr or Ser <220> FEATURE: <222> LOCATION: 48<223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il #e, Asn or Gln<220> FEATURE: <222> LOCATION: 53<223> OTHER INFORMATION: Arg, His or Lys <220> FEATURE:<222> LOCATION: 54 <223> OTHER INFORMATION: Asp or Glu <220> FEATURE:<222> LOCATION: 71 <223> OTHER INFORMATION: Ser or Thr <220> FEATURE:<222> LOCATION: 79 <223> OTHER INFORMATION: Glu, Asp, Gln or Asn<220> FEATURE: <222> LOCATION: 83 <223> OTHER INFORMATION: Glu or Asp<220> FEATURE: <222> LOCATION: 84<223> OTHER INFORMATION: Arg, His or Lys <220> FEATURE:<222> LOCATION: 85 <223> OTHER INFORMATION: Gly, Ala, Val, Leu or  #Ile<220> FEATURE: <222> LOCATION: 87<223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il #e, Thr or Ser<220> FEATURE: <222> LOCATION: 95<223> OTHER INFORMATION: Met, Cys, Gln, Asn, Ar #g, Lys or His<220> FEATURE: <222> LOCATION: 100<223> OTHER INFORMATION: Arg, His or Lys <220> FEATURE:<222> LOCATION: 107 <223> OTHER INFORMATION: Trp, Phe, Tyr, Arg, Hi#s or Lys <220> FEATURE: <222> LOCATION: 114<223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il #e, Ser, Thr, Tyr or Phe<220> FEATURE: <222> LOCATION: 115<223> OTHER INFORMATION: Gln, Asn, Asp or Glu <220> FEATURE:<222> LOCATION: 116 <223> OTHER INFORMATION: Asp or Glu <220> FEATURE:<222> LOCATION: 125 <223> OTHER INFORMATION: Gly, Ala, Val, Leu, or# Ile <220> FEATURE: <222> LOCATION: 134<223> OTHER INFORMATION: Arg, His or Lys <220> FEATURE:<222> LOCATION: 135 <223> OTHER INFORMATION: Asn, Gln, Thr or Ser<220> FEATURE: <222> LOCATION: 139<223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il #e, Ser, Thr, Met or Cys<220> FEATURE: <222> LOCATION: 141<223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il #e, Thr or Ser<220> FEATURE: <222> LOCATION: 157<223> OTHER INFORMATION: Arg, His or Lys <220> FEATURE:<222> LOCATION: 158 <223> OTHER INFORMATION: Asn, Gln, Gly, Ala, Va#l, Leu or Ile <220> FEATURE: <222> LOCATION: 160<223> OTHER INFORMATION: Gly, Ala, Val, Leu or  #Ile <220> FEATURE:<222> LOCATION: 162 <223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il#e, Ser, Thr or Cys <220> FEATURE: <222> LOCATION: 166<223> OTHER INFORMATION: Gly, Ala, Val, Leu, Il #e, Thr or Ser<220> FEATURE: <222> LOCATION: 167 <223> OTHER INFORMATION: Asp or Glu<400> SEQUENCE: 22 Cys Gly Pro Gly Arg Gly Xaa Xaa Xaa Arg Ar#g Xaa Xaa Xaa Pro Lys   1               5  #                 10 #                 15 Xaa Leu Xaa Pro Leu Xaa Tyr Lys Gln Phe Xa#a Pro Xaa Xaa Xaa Glu              20      #             25     #             30 Xaa Thr Leu Gly Ala Ser Gly Xaa Xaa Glu Gl#y Xaa Xaa Xaa Arg Xaa          35          #         40         #         45 Ser Glu Arg Phe Xaa Xaa Leu Thr Pro Asn Ty#r Asn Pro Asp Ile Ile      50              #     55             #     60 Phe Lys Asp Glu Glu Asn Xaa Gly Ala Asp Ar#g Leu Met Thr Xaa Arg  65                  # 70                 # 75                  # 80 Cys Lys Xaa Xaa Xaa Asn Xaa Leu Ala Ile Se#r Val Met Asn Xaa Trp                  85  #                 90 #                 95 Pro Gly Val Xaa Leu Arg Val Thr Glu Gly Xa#a Asp Glu Asp Gly His             100       #           105      #           110 His Xaa Xaa Xaa Ser Leu His Tyr Glu Gly Ar#g Ala Xaa Asp Ile Thr         115           #       120          #       125 Thr Ser Asp Arg Asp Xaa Xaa Lys Tyr Gly Xa#a Leu Xaa Arg Leu Ala     130               #   135              #   140 Val Glu Ala Gly Phe Asp Trp Val Tyr Tyr Gl#u Ser Xaa Xaa His Xaa 145                 1 #50                 1#55                 1 #60 His Xaa Ser Val Lys Xaa Xaa                165 <210> SEQ ID NO 23 <211> LENGTH: 74 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: primer<400> SEQUENCE: 23gcgcgcttcg aagcgaggca gccagcgagg gagagagcga gcgggcgagc cg#gagcgagg     60 aaatcgatgc gcgc               #                  #                   #     74 <210> SEQ ID NO 24 <211> LENGTH: 74<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: primer<400> SEQUENCE: 24gcgcgcagat ctgggaaagc gcaagagaga gcgcacacgc acacacccgc cg#cgcgcact     60 cgggatccgc gcgc               #                  #                   #     74 <210> SEQ ID NO 25 <211> LENGTH: 996<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: gene      activation construct <400> SEQUENCE: 25cgaagcgagg cagccagcga gggagagagc gagcgggcga gccggagcga gg#aaatcgaa     60ggttcgaatc cttcccccac caccatcact ttcaaaagtc cgaaagaatc tg#ctccctgc    120ttgtgtgttg gaggtcgctg agtagtgcgc gagtaaaatt taagctacaa ca#aggcaagg    180cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tg#cttcgcga    240tgtacgggcc agatatacgc gttgacattg attattgact agttattaat ag#taatcaat    300tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac tt#acggtaaa    360tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tg#acgtatgt    420tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggact at#ttacggta    480aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ct#attgacgt    540caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gg#gactttcc    600tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc gg#ttttggca    660gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tc#caccccat    720tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aa#tgtcgtaa    780caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tc#tatataag    840cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa tt#aatacgac    900tcactatagg gagacccaag cttggtaccg agctcggatc gatctgggaa ag#cgcaagag    960 agagcgcaca cgcacacacc cgccgcgcgc actcgg      #                   #      996 <210> SEQ ID NO 26 <211> LENGTH: 26<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: antisense      construct <400> SEQUENCE: 26gtcctggcgc cgccgccgcc gtcgcc           #                  #              26 <210> SEQ ID NO 27 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: antisense      construct <400> SEQUENCE: 27ttccgatgac cggcctttcg cggtga           #                  #              26 <210> SEQ ID NO 28 <211> LENGTH: 26 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: antisense      construct <400> SEQUENCE: 28gtgcacggaa aggtgcaggc cacact           #                  #              26

I claim:
 1. A method for promoting growth of hair on skin of a mammal,comprising administering an agent to the mammal for a time and underconditions effective to promote growth of hair on the skin of themammal, wherein the agent is a hedgehog agonist or a patched antagonistthat promotes hedgehog signal transduction, and wherein said hedgehogagonist or patched antagonist promotes hedgehog signal transduction by amechanism selected from (i) binding to patched; (ii) altering thebinding or enzymatic activity of a protein involved in hedgehog signaltransduction; or (iii) altering expression of a hedgehog protein, apatched protein or a protein involved in hedgehog signal transduction.2. The method of claim 1, wherein the agent is a patched antagonist. 3.The method of claim 2, wherein the patched antagonist binds to patchedand mimics hedgehog-mediated patched signal transduction.
 4. The methodof claim 3, wherein the binding of the patched antagonist to patchedresults in upregulation of patched and/or glioma-associated oncogenehomolog (gli) expression.
 5. The method of claim 2, wherein the patchedantagonist alters the level of expression of a hedgehog protein, apatched protein or a protein involved in the intracellular signaltransduction pathway of patched.
 6. The method of claim 1, wherein theagent is a hedgehog agonist.
 7. The method of claim 6, wherein thehedgehog agonist is a peptidomimetic of a hedgehog polypeptide sequence.8. The method of claim 6, wherein the hedgehog agonist is a polypeptideincluding a hedgehog polypeptide sequence of at least a bioactiveextracellular portion of a hedgehog protein.
 9. The method of claim 8,wherein the polypeptide includes at least 50 contiguous amino acidsresidues of an N-terminal half of the hedgehog protein.
 10. The methodof claim 8, wherein the polypeptide includes at least 100 contiguousamino acids of an extracellular domain of the hedgehog protein.
 11. Themethod of claim 8, wherein the polypeptide includes at least a 19 kdfragment of an extracellular domain of the hedgehog protein.
 12. Themethod of claim 8, wherein the hedgehog protein is encoded by a gene ofa vertebrate organism.
 13. The method of claim 8, wherein thepolypeptide includes a hedgehog polypeptide sequence represented in SEQID No:
 21. 14. The method of claim 13, wherein the hedgehog polypeptidesequence is an amino acid sequence of a hedgehog protein selected fromthe group consisting of SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ IDNo:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15 and SEQ ID No:16. 15.The method of claim 8, wherein the polypeptide includes a hedgehogpolypeptide sequence represented in SEQ ID No:
 22. 16. The method ofclaim 8, wherein the hedgehog protein is encoded by a human hedgehoggene.
 17. The method of claim 8, wherein the hedgehog polypeptidesequence is at least 60 percent identical to an amino acid sequence of ahedgehog protein selected from the group consisting of SEQ ID No:9, SEQID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13, SEQ ID No:14, SEQ IDNo:15 and SEQ ID No:16.
 18. The method of claim 17, wherein the hedgehogpolypeptide sequence is at least 75 percent identical to an amino acidsequence of a hedgehog protein selected from the group consisting of SEQID No:9, SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID No:13, SEQ IDNo:14, SEQ ID No:15 and SEQ ID No:16.
 19. The method of claim 17,wherein the hedgehog polypeptide sequence is at least 85 percentidentical to an amino acid sequence of a hedgehog protein selected fromthe group consisting of SEQ ID No:9, SEQ ID No:10, SEQ ID No:11, SEQ IDNo:12, SEQ ID No:13, SEQ ID No:14, SEQ ID No:15 and SEQ ID No:16. 20.The method of claim 8, wherein the hedgehog polypeptide sequence is anamino acid sequence of a Sonic hedgehog protein.
 21. The method of claim8, wherein the hedgehog polypeptide sequence is an amino acid sequenceof a Desert hedgehog protein.
 22. The method of claim 8, wherein thehedgehog polypeptide sequence is an amino acid sequence of an Indianhedgehog protein.
 23. The method of claim 8, wherein the hedgehogpolypeptide sequence includes amino acid residues 24-193 of SEQ ID No:15.
 24. The method of claim 8, wherein the polypeptide is purified to atleast 80% by dry weight.
 25. The method of claim 8, wherein thepolypeptide is a recombinantly produced polypeptide.
 26. The method ofclaim 8, wherein the polypeptide is a chemically synthesizedpolypeptide.
 27. A method for promoting growth of hair on skin of amammal, comprising administering a composition to the mammal for a timeand under conditions effective to promote growth of hair on the skin ofthe mammal, wherein said composition comprises an agent and anacceptable carrier or excipient, and wherein the agent is a hedgehogagonist or a patched antagonist that promotes hedgehog signaltransduction, and further said hedgehog agonist or patched antagonistpromotes hedgehog signal transduction by a mechanism selected from (i)binding to patched; (ii) altering the binding or enzymatic activity of aprotein involved in hedgehog signal transduction; or (iii) alteringexpression of a hedgehog protein, a patched protein or a proteininvolved in hedgehog signal transduction.
 28. The method of claim 27,wherein the composition is applied topically.